CN109575142B - 一种cd4辅助性t细胞表位融合肽及其疫苗 - Google Patents

一种cd4辅助性t细胞表位融合肽及其疫苗 Download PDF

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CN109575142B
CN109575142B CN201811138035.6A CN201811138035A CN109575142B CN 109575142 B CN109575142 B CN 109575142B CN 201811138035 A CN201811138035 A CN 201811138035A CN 109575142 B CN109575142 B CN 109575142B
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徐建青
黄杨
张晓燕
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Vacdiagn Biotechnology Co ltd
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Abstract

本发明提供了一种CD4辅助性T细胞表位融合肽、其编码核酸以及包含其的免疫组合物。所述表位融合肽包含巨细胞病毒表位和/或流感病毒表位。本发明提供的表位融合肽能大幅度提高目的免疫原，尤其是弱免疫原的细胞免疫应答水平，是克服免疫系统对抗原免疫耐受性，尤其是肿瘤抗原或感染相关抗原的有效手段，适用于高效增强疫苗的效力。

Description

一种CD4辅助性T细胞表位融合肽及其疫苗
技术领域
本发明属于分子生物学和免疫学领域。具体地，本发明涉及一种CD4辅助性T细胞表位融合肽，尤其涉及包含该表位融合肽的疫苗及其应用。
背景技术
T辅助细胞(Th细胞)是一种在免疫系统中起重要作用的T细胞，特别是在适应性免疫系统中。它们通过释放T细胞细胞因子来帮助其他免疫细胞的活性。这些细胞有助于抑制或调节免疫反应。它们在B细胞抗体类别的转换、细胞毒性T细胞的活化和生长以及吞噬细胞如巨噬细胞的杀菌活性最大化中是必需的。
成熟Th细胞表达蛋白CD4，称为CD4+T细胞。通常将这样的CD4+T细胞作为免疫系统内的辅助T细胞进行预定义的处理。例如，当抗原呈递细胞在II类MHC上表达抗原时，CD4+细胞将通过细胞与细胞相互作用的组合(例如CD40(蛋白质)和CD40L)和细胞因子来帮助这些细胞。
可以从HIV(一种主要感染CD4+T细胞的病毒)中看到辅助T细胞的重要性。在艾滋病毒感染的晚期阶段，功能性CD4+T细胞的丧失导致被称为获得性免疫缺陷综合征(AIDS)的感染症状阶段。当血液或其他体液早期发现HIV病毒时，连续治疗可能会延迟发生这种情况的时间。如果发生艾滋病，治疗也可以更好地管理艾滋病的过程。还有其他罕见的疾病，如淋巴细胞减少症，导致CD4+T细胞的缺失或功能障碍。这些疾病产生类似的症状，其中许多是致命的。
抗原表位(antigenic epitope)，简称“表位”(epitope)，也称为“抗原决定簇”(antigenic determinant)，是指抗原表面上决定抗原特异性的化学基团。抗原表位可被免疫系统(尤其是抗体、B细胞或者T细胞)所识别。抗体中能识别抗原表位的区域叫做“互补位”或“抗体决定簇”。尽管通常抗原表位是指外来蛋白质等物质的其中一部分，但只要能被自身免疫系统所识别的表位，也被归为抗原表位。
蛋白质抗原的表位根据它们的结构以及与互补位的交互作用，被分为构象表位和线性表位这两种类型。其中构象表位由抗原氨基酸序列中的不连续部分组成，因此互补位和抗原表位的交互作用是基于表面的三维特征和形状，或者是抗原的三级结构。大部分的抗原表位都属于构象表位。与此相反，线性表位是由一段连续的抗原氨基酸序列构成，与抗原的交互作用的基础是其一级结构。
T细胞表位(T cell epitope)主要是由8-17氨基酸组成的短肽构成，出现在抗原呈递细胞(antigen-presenting cell,APC)上，这种抗原表位将会和主要组织相容性复合体(MHC)相结合形成复合体，并与相应的T细胞表位受体结合，从而活化T细胞，产生相应细胞免疫应答(Shimonkevitz et al.,1984；Babbitt et al.,1985；Buus et al.,1986；Townsend and Bodmer,1989)。与表位结合的MHC主要有两类分子，其中，I类主要组织相容性复合体所呈现的T细胞抗原表位通常是由8至11个氨基酸长度的多肽组成，而II类主要组织相容性复合体所呈现的T细胞抗原表位相对更长，由13-17个氨基酸组成。
辅助T细胞表位(Th epitope，即Th表位)是指T细胞表位中的，与MHC分子结合形成的复合体能被CD4辅助性T细胞受体识别的一类T细胞表位。Th表位主要与存在于由主要组织相容性复合体(MHC)的II类基因编码的抗原呈递细胞(antigen-presenting cell,APC)表面上的分子结合。然后，II类分子和肽表位的复合物被T辅助淋巴细胞表面上的特异性T细胞受体(TCR)识别。以这种方式，在MHC分子的环境中呈现抗原表位的T细胞可以被激活，并为B淋巴细胞分化提供必要的信号。传统上，肽免疫原的辅助T细胞表位的来源是肽共价偶联的载体蛋白，但是该偶联过程可引入其他问题，例如在偶联过程中抗原决定簇的修饰和以针对肽的抗体为代价的针对载体的抗体的诱导(Schutze,M.P.,Leclerc,C.Jolivet,M.Audibert,F.Chedid,L.Carrier-induced epitopic suppression,a major issue forfuture synthetic vaccines.J Immunol.1985,135,2319-2322；DiJohn,D.,Torrese,J.R.Murillo,J.Herrington,D.A.et al.Effect of priming with carrier on responseto conjugate vaccine.The Lancet.1989,2,1415-1416)。此外，在制备中使用无关蛋白引入质量控制问题。合适的载体蛋白的选择在设计肽疫苗时非常重要，其选择受到其大规模生产的毒性和可行性等因素的限制。这种方法还有其他限制，包括可偶联的肽负载的大小和可以安全施用的载体的剂量(Audibert,F.a.C.,L.1984.Modern approaches tovaccines.Molecular and chemical basis of virus virulence and immunogenicity.,Cold Spring Harbor Laboratory,New York.)。虽然载体分子允许诱导强的免疫应答，但它们也与不利影响相关，例如抑制抗肽抗体应答(Herzenberg,L.A.and Tokuhisa,T.1980.Carrier-priming leads to hapten-specific suppression.Nature 285:664；Schutze,M.P.,Leclerc,C.,Jolivet,M.,Audibert,F.,and Chedid,L.1985.Carrier-induced epitopic suppression,a major issue for future synthetic vaccines.JImmunol 135:2319；Etlinger,H.M.,Felix,A.M.,Gillessen,D.,Heimer,E.P.,Just,M.,Pink,J.R.,Sinigaglia,F.,Sturchler,D.,Takacs,B.,Trzeciak,A.,and et al.,1988.Assessment in humans of a synthetic peptide-based vaccine against thesporozoite stage of the human malaria parasite,Plasmodium falciparum.JImmunol 140:626)。
一般来说，免疫原必须包含辅助T细胞表位，除了将被表面Ig识别的表位或存在于细胞毒性T细胞上的受体之外。应该意识到，这些类型的表位可能是非常不同的。对于B细胞表位，构象是重要的，因为B细胞受体直接与天然免疫原结合。相比之下，由T细胞识别的表位不依赖于表位的构象完整性，并且由针对CTL的约9个氨基酸的短序列和针对辅助T细胞的稍长的序列(长度限制较小)组成。这些表位的唯一要求是它们可以分别容纳在I类或II类分子的结合裂口中，并且然后复合物能够与T细胞受体结合。II类分子的结合位点在两端是开放的，允许与报道的短至8个氨基酸残基(Fahrer,A.M.,Geysen,H.M.,White,D.O.,Jackson,D.C.and Brown,L.E.Analysis of the requirements for class II-restricted T-cell recognition of a single determinant reveals considerablediversity in the T-cell response and degeneracy of peptide binding to HEdJ.Immunol.1995.155:2849-2857)的表位结合的肽的长度变化更大(Brown,J.H.,T.S.Jardetzky,J.C.Gorga,L.J.Stern,R.G.Urban,J.L.Strominger andD.C.Wiley.1993.Three-dimensional structure of the human class IIhistocompatibility antigen HLA-DR1.Nature 364:33)。
Th表位能刺激并活化辅助T细胞，相应地，促进CD8T细胞和B细胞活化，最终提高免疫应答。实质上，Th表位除了能活化针对自身的免疫应答外，还能有效帮助与之关联的其他抗原或表位的免疫应答。因此，可以将异源性的强Th表位与目的免疫原融合，据此可以提高目的免疫原的免疫原性。一种称之为“PADRE(pan HLA DR-binding Epitope))”的人造强Th表位已用于多个疫苗的融合构造，以提高相关免疫原的免疫应答水平(del Guercio etal.,Vaccine,1997,15:441.；Franke,E.D.et al.,Vaccine,1999,17:1201；JeffAlexander et al.,J Immunol,2000,164(3)1625-1633；Jeff Alexander et al.,Vaccine,2004,22:2362.；La Rosa,Corinna et al.,The Journal of infectiousdiseases,2012,205:1294-304)。此外，源于破伤风毒素(Tetanus toxin)的强Th表位P2也常用于与目的免疫原偶联，以提高免疫原性(Panina-Bordignon P et al.,Eur JImmunol,1989,19:2237-42；La Rosa,Corinna et al.,The Journal of infectiousdiseases,2012,205:1294-304)。
但一般而言，用于提高免疫原性的Th表位通常为异源性的，换言之，疫苗受试者体内不会存在针对该Th表位自身的高水平的免疫应答。因此，使用上述的强Th表位接种疫苗受试者时，很可能的情况是疫苗受试者的免疫系统是初次接触此类Th表位，受体免疫系统活化针对此类Th表位和目的免疫原的表位基本上是同步的，针对此类Th表位的T细胞的生成时间和数量与目的免疫原相似，这样的话，对于帮助目的免疫原的效果会因此受限。特别是对于弱免疫原性的肿瘤抗原，此类Th表位的辅助作用更难发挥。事实上，直接采用强Th表位虽然能活化肿瘤抗原，但其激发的细胞免疫应答水平仍较低，不能满足肿瘤疫苗的需要(Ghaffari-Nazari H et al.,PLoS ONE,2015,10(11):e0142563)。
因此，需要新的Th表位策略来提高目的免疫原免疫原性，特别是一些弱的免疫原，例如肿瘤抗原。
发明内容
本发明的目的是提供一种CD4辅助性T细胞表位融合肽，通过该表位融合肽，目的免疫原的免疫原性得以提高。
进一步地，本发明利用来源于巨细胞病毒(Cytomegalovirus，CMV)和流感病毒(Influvirus,Flu)的强Th表位获得表位融合肽来提高目的免疫原的免疫原性。
为了本发明的目的以下定义下列术语。
“表位融合肽”是指将若干表位连接在一起所形成的一个肽。
“目的免疫原”是指为实现某种免疫应答，所采用的免疫原，包括抗原等具有免疫学活性的物质，优选为蛋白。
本发明的另一目的是提供一种所述表位融合肽和所述目的免疫原的融合蛋白。
为实现上述目的，本发明提供一种CD4辅助性T细胞表位融合肽，其包含巨细胞病毒表位和/或流感病毒表位。
在本发明的一个实施方案中，所述表位融合肽包含选自SEQ ID NO：1-10所示的巨细胞病毒表位中的一种或多种，和/或选自SEQ ID NO：11-23所示的流感病毒表位中的一种或多种。
在本发明的一个实施方案中，所述表位融合肽由选自SEQ ID NO：1-10所示的巨细胞病毒表位中的一种或多种，和/或选自SEQ ID NO：11-23所示的流感病毒表位中的一种或多种组成。优选地，所述表位融合肽由5个或10个巨细胞病毒表位和/或由8个或13个流感病毒表位组成，例如SEQ ID NO:34或44所示的表位融合肽。最优选地，所述表位融合肽由13个流感病毒表位组成，例如SEQ ID NO:48所示的表位融合肽。
优选地，所述表位融合肽诱导体液或细胞免疫应答。
本发明还提供了所述表位融合肽和目的免疫原的融合蛋白。
本发明还提供了编码所述表位融合肽和/或所述融合蛋白的多核苷酸。
在本发明的一个实施方案中，所述目的免疫原为任意一种或多种免疫原。优选的，所述目的免疫原为肽、抗原、半抗原、碳水化合物、蛋白质、核酸、过敏原、病毒或病毒的一部分、细菌、寄生虫或其它完整的微生物。
在本发明的一个实施方案中，所述抗原为肿瘤抗原或感染相关抗原。
在本发明的一个实施方案中，所述肿瘤抗原选自肺癌抗原、睾丸癌抗原、黑色素瘤抗原、肝癌抗原、乳腺癌抗原或前列腺癌抗原中的一种或多种。
在本发明的一个实施方案中，所述肿瘤抗原选自LAGE抗原、MAGE抗原或NY-ESO-1抗原中的一种或多种。优选地，LAGE抗原为LAGE-1，MAGE抗原为MAGE-A3。进一步优选地，所述肿瘤抗原包含LAGE-1、MAGE-A3和NY-ESO-1。优选地，所述LAGE-1的氨基酸序列如SEQ IDNO:24所示，所述MAGE-A3的氨基酸序列如SEQ ID NO:25所示，所述NY-ESO-1的氨基酸序列如SEQ ID NO:26所示。
在本发明的一个实施方案中，所述感染相关抗原选自HIV抗原、流感病毒抗原或HBV抗原中的一种或多种。
优选地，所述融合蛋白如SEQ ID NO：55-58中之一所示。
本发明的另一目的是提供一种免疫组合物，所述免疫组合物包含治疗有效量的根据本发明的表位融合肽、融合蛋白和/或多核苷酸，以及药学上可接受的载体。优选地，该免疫组合物为疫苗。
本发明的另一目的是提供一种药盒，所述药盒包含根据本发明的表位融合肽、融合蛋白、多核苷酸和/或免疫组合物，以及其使用说明。
本发明还提供了根据本发明的表位融合肽、融合蛋白、多核苷酸和/或免疫组合物在制备提高目的免疫原的免疫原性的药物或疫苗中用途。
本发明还提供了一种使用根据本发明的表位融合肽提高目的免疫原的免疫原性的方法，包括将疫苗受试者或人群中已有较强免疫应答的CD4辅助性T细胞表位与目的免疫原融合形成的融合蛋白。其方法具体为：
(1)选择一个或多个CD4辅助性T细胞的表位，所述表位与MHC分子结合形成的复合体能被CD4辅助性T细胞受体识别，并且在疫苗接种前，疫苗受试者体内已经存在针对所述表位中至少一个表位产生了T细胞免疫应答；
(2)将上述表位融合制成表位融合肽，将所述表位融合肽与目的免疫原融合制成融合蛋白，表达融合蛋白并制成疫苗，其表达载体形式可以是DNA疫苗载体形式，可以是蛋白疫苗载体形式，还可以是病毒疫苗载体形式；
(3)将上述疫苗接种给疫苗受试者，接种时可以选择适当的佐剂，例如不完全弗氏佐剂，完全弗氏佐剂，氢氧化铝佐剂等。
进一步地，所述使用方法的步骤(1)还包括检查疫苗受试者MHC表型的步骤。优选地，检查疫苗受试者MHC表型包括检查疫苗受试者的MHCII类基因亚型型别。
本发明还提供了一种治疗或预防有需要的受试者的病症的方法，其包括给予治疗有效量的本发明的表位融合肽、融合蛋白、免疫组合物和/或多核苷酸。优选地，所述病症选自恶性肿瘤、细菌和病毒性慢性感染中的一种或多种。优选地，所述恶性肿瘤为乳腺癌或结肠癌。优选地，在该方法中，初免使用DNA疫苗载体，加强免疫使用蛋白疫苗载体，更优选地，初免使用pVKD1.0-CI-LMNB DNA疫苗，加强免疫使用LMNB-I13蛋白。
本发明提供的表位融合肽能大幅度提高目的免疫原，尤其是弱免疫原的细胞免疫应答水平，是克服免疫系统对抗原免疫耐受性，尤其是对肿瘤抗原或感染相关抗原免疫耐受性的有效手段，适用于高效增强疫苗的效力。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1和图2分别是带有LAGE-1、MAGE-A3和NY-ESO-1抗原编码序列的DNA疫苗载体pVKD1.0-hLMN的质粒图谱和双酶切鉴定图。
图3和图4分别是带有LAGE-1、MAGE-A3和NY-ESO-1抗原以及霍乱毒素B亚单位编码序列的DNA疫苗载体pVKD1.0-hLMN-CTB的质粒图谱和双酶切鉴定图。
图5和图6分别是带有源于CMV和流感病毒CD4表位编码序列的DNA疫苗载体pVKD1.0-CI的质粒图谱和双酶切鉴定图。
图7和图8分别是带有LAGE-1、MAGE-A3和NY-ESO-1抗原以及霍乱毒素B亚单位，和源于CMV和流感病毒CD4表位编码序列的DNA疫苗载体pVKD1.0-CI-LMNB的质粒图谱和双酶切鉴定图。
图9和图10分别是带有LAGE-1、MAGE-A3和NY-ESO-1抗原编码序列的原核载体pET-30a(+)-LMN的质粒图谱和双酶切鉴定图。
图11和图12分别是带有LAGE-1、MAGE-A3和NY-ESO-1抗原以及霍乱毒素B亚单位编码序列的原核载体pET-30a(+)-LMN-CTB的质粒图谱和双酶切鉴定图。
图13和图14分别是带有源于CMV表位编码序列的原核载体pET-30a(+)-CMV Th的质粒图谱和双酶切鉴定图。
图15和图16分别是带有源于CMV表位和LAGE-1、MAGE-A3和NY-ESO-1抗原以及霍乱毒素B亚单位编码序列的原核载体pET-30a(+)-CMV10-LMNB的质粒图谱和双酶切鉴定图。
图17和图18分别是带有源于流感病毒表位编码序列的原核载体pET-30a(+)-CMVTh的质粒图谱和双酶切鉴定图。
图19和图20分别是带有源于流感病毒表位和LAGE-1、MAGE-A3和NY-ESO-1抗原以及霍乱毒素B亚单位编码序列的原核载体pET-30a(+)-Influ8-LMNB的质粒图谱和双酶切鉴定图。
图21和图22分别是带有源于流感病毒表位和LAGE-1、MAGE-A3和NY-ESO-1抗原以及霍乱毒素B亚单位编码序列的原核载体pET-30a(+)-Influ13-LMNB的质粒图谱和双酶切鉴定图。
图23为动物免疫实验中的细胞免疫应答检测结果。
图24和图25分别是带有源于CMV表位和LAGE-1、MAGE-A3和NY-ESO-1抗原以及霍乱毒素B亚单位编码序列的原核载体pET-30a(+)-CMV5-LMNB的质粒图谱和双酶切鉴定图。
图26为实施例12中的动物免疫实验中的细胞免疫应答检测结果。
图27为实施例13中的小鼠肿瘤生长情况。
图28和图29分别是实施例13中的小鼠无瘤存活状况和总体存活状况。
图30为4T1-hNY-ESO-1小鼠肿瘤模型中，各治疗小组小鼠肿瘤生长情况。
图31为CT26-hLAGE-1小鼠肿瘤模型中，各治疗小组小鼠肿瘤生长情况。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。
实施例1DNA疫苗pVKD1.0-hLMN构建
LAGE-1、MAGE-A3和NY-ESO-1氨基酸序列分别如SEQ ID NO:24-26所示。通过在线密码子优化软件(http://www.jcat.de/)将上述抗原氨基酸序列优化成哺乳动物密码子使用偏好的核苷酸序列，分别如SEQ ID NO:27-29所示。经上海捷瑞生物技术有限公司合成后，通过本领域熟知的方法克隆至DNA疫苗载体pVKD1.0(苏州工业园区唯可达生物科技有限公司提供)上的多克隆位点Sal I与BamH I之间，构建成可表达融合蛋白抗原的DNA疫苗载体pVKD1.0-hLMN(质粒图谱如图1)，经测序鉴定正确后入库。用限制内切酶Sal I与BamHI鉴定载体pVKD1.0-hLMN(酶切体系如表1)，其酶切验证图谱如图2所示。
表1：质粒pVKD1.0-hLMN的酶切鉴定体系(37℃酶切2小时)
酶切体系 体积
质粒pVKD1.0-hLMN 3μL，约1μg
Sal I(宝生物，货号1080A) 1μL
BamH I(宝生物，货号1010A) 1μL
酶切缓冲液 1μL
ddH<sub>2</sub>O 补至10μL
实施例2DNA疫苗pVKD1.0-hLMN-CTB构建
霍乱毒素B亚单位(Cholera toxin subunit B，简称CTB)的氨基酸序列(SEQ IDNO:30)的哺乳动物密码子优化序列(SEQ ID NO:31)及其真核表达载体pVKD1.0-CTB由苏州工业园区唯可达生物科技有限公司提供。以pVKD1.0-CTB为模板，设计引物(见表2)，通过PCR扩增CTB基因片段，然后凝胶回收相应片段，通过同源重组法将CTB片段插入线性化载体pVKD1.0-hLMN相应位置上，构建成DNA疫苗载体pVKD1.0-hLMN-CTB(质粒图谱如图3)，经测序鉴定正确后入库。用限制内切酶Sal I与BamH I鉴定载体pVKD1.0-hLMN-CTB(酶切体系如表3)，其酶切验证图谱如图4所示。
表2：实施例2中的引物
Figure BDA0001815105190000081
表3：质粒pVKD1.0-hLMN-CTB的酶切鉴定体系(37℃酶切2小时)
酶切体系 体积
质粒pVKD1.0-hLMN-CTB 3μL，约1μg
Sal I(宝生物，货号1080A) 1μL
BamH I(宝生物，货号1010A) 1μL
酶切缓冲液 1μL
ddH<sub>2</sub>O 补至10μL
实施例3DNA疫苗pVKD1.0-CI-LMNB构建
在免疫表位数据库(IEDB，http://www.iedb.org)上获取来源于巨细胞病毒(Cytomegalovirus，CMV)和流感病毒(Influvirus,Flu)的强Th表位(见表4)，其中，CMV的强Th表位包括pp65-11、pp65-71、pp65-92、pp65-123、pp65-128、pp65-57、pp65-62、pp65-30、pp65-112和pp65-104；流感病毒的强Th表位包括HA203、NP438、NS1-84、M1-181、HA375、NP24、NP95、NP221、HA434、HA440、NP324、M1-127和M1-210。表4中选择的表位覆盖人群中MHCII类分子多数亚型，而且也覆盖小鼠MHC II亚型分子。然后将所选的表位pp65-11、pp65-71、pp65-92、pp65-123、pp65-128、HA203、NP438、NS1-84、M1-181、HA375、NP24、NP95、NP221串联在一起，形成一段CMV病毒和流感病毒的表位融合肽，其氨基酸序列如SEQ ID NO:34所示，该表位融合肽经哺乳动物密码子优化后，核酸序列如SEQ ID NO:35所示。送苏州泓迅生物科技有限公司合成该核酸序列，然后通过本领域熟知的分子生物学方法将该核酸序列插入至DNA疫苗载体pVKD1.0(苏州工业园区唯可达生物科技有限公司)上，形成载体pVKD1.0-CI(质粒图谱如图5)，经测序鉴定正确后入库。用限制内切酶Pst I与Bgl II鉴定载体pVKD1.0-CI(酶切体系如表5)，其酶切验证谱如图6所示。
表4：实施例3中的Th表位
表位名称 来源 氨基酸序列
pp65-11 CMV LLQTGIHVRVSQPSL(SEQ ID NO:1)
pp65-71 CMV IIKPGKISHIMLDVA(SEQ ID NO:2)
pp65-92 CMV EHPTFTSQYRIQGKL(SEQ ID NO:3)
pp65-123 CMV AGILARNLVPMVATV(SEQ ID NO:4)
pp65-128 CMV KYQEFFWDANDIYRI(SEQ ID NO:5)
pp65-57 CMV KVYLESFCEDVPSGK(SEQ ID NO:6)
pp65-62 CMV TLGSDVEEDLTMTRN(SEQ ID NO:7)
pp65-30 CMV PLKMLNIPSINVHHY(SEQ ID NO:8)
pp65-112 CMV ACTSGVMTRGRLKAE(SEQ ID NO:9)
pp65-104 CMV TERKTPRVTGGGAMA(SEQ ID NO:10)
HA203 Influ NQRALYHTENAYVSVVS(SEQ ID NO:11)
NP438 Influ SDMRAEIIKMMESARPE(SEQ ID NO:12)
NS1-84 Influ ALASRYLTDMTIEEMSR(SEQ ID NO:13)
M1-181 Influ LASTTAKAMEQMAGSSE(SEQ ID NO:14)
HA375 Influ SGYAADQKSTQNAINGITNKVN(SEQ ID NO:15)
NP24 Influ EIRASVGKMIDGIGRFYI(SEQ ID NO:16)
NP95 Influ PIYRRVDGKWMRELVLY(SEQ ID NO:17)
NP221 Influ RMCNILKGKFQTAAQRAM(SEQ ID NO:18)
HA434 Influ IWTYNAELLVLLENERT(SEQ ID NO:19)
HA440 Influ ELLVLLENERTLDFHDS(SEQ ID NO:20)
NP324 Influ HKSQLVWMACNSAAFED(SEQ ID NO:21)
M1-127 Influ CMGLIYNRMGAVTTESA(SEQ ID NO:22)
M1-210 Influ RQMVQAMRAIGTHPSSSTGLKND(SEQ ID NO:23)
表5：质粒pVKD1.0-CI的酶切鉴定体系(37℃酶切2小时)
酶切体系 体积
质粒pVKD1.0-CI 3μL，约1μg
Pst I(宝生物，货号1073A) 1μL
Bgl II(宝生物，货号1021A) 1μL
酶切缓冲液 1μL
ddH<sub>2</sub>O 补至10μL
最后以实施例2中的载体pVKD1.0-hLMN-CTB为模板，设计引物(见表6)，通过PCR扩增hLMN-CTB目的基因片段，然后通过本领域熟知的分子生物学方法将该基因片段插入该pVKD1.0-CI载体上Not I和Bam HI酶切位点之间，构建成DNA疫苗载体pVKD1.0-CI-LMNB(质粒图谱如图7)，经测序鉴定正确后入库。用限制内切酶Bam HI与EcoR V鉴定载体pVKD1.0-CI-LMNB(酶切体系如表7)，其酶切验证图谱如图8所示。
表6：实施例3中的引物
Figure BDA0001815105190000111
表7：质粒pVKD1.0-CI-LMNB的酶切鉴定体系(37℃酶切2小时)
酶切体系 体积
质粒pVKD1.0-CI-LMNB 3μL，约1μg
Bam HI(宝生物，货号1010A) 1μL
EcoR V(宝生物，货号1042A) 1μL
酶切缓冲液 1μL
ddH<sub>2</sub>O 补至10μL
实施例4LMN原核表达载体构建
LAGE-1、MAGE-A3和NY-ESO-1氨基酸序列分别如SEQ ID NO:24-26所示。通过在线密码子优化软件(http://www.jcat.de/)将所述抗原氨基酸序列优化成大肠杆菌密码子使用偏好的核苷酸序列，其核苷酸序列分别如SEQ ID NO:38-40所示。经苏州泓迅生物科技有限公司合成后，通过本领域熟知的分子生物学方法插入至原核表达载体pET-30a(+)(Novagen，货号69909)上的多克隆位点Nco I与Xho I之间，构建成原核表达构建体pET-30a(+)-LMN(质粒图谱如图9)，经测序鉴定正确后入库。用限制内切酶Nco I和Xho I鉴定载体pET-30a(+)-LMN(酶切体系如表8)，其酶切验证图谱如图10所示。
表8：质粒pET-30a(+)-LMN的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-LMN 3μL，约1μg
Nco I(宝生物，货号1160A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH<sub>2</sub>O 补至10μL
实施例5LMN-CTB原核表达载体构建
霍乱毒素B亚单位CTB氨基酸序列(SEQ ID NO:30)及其原核密码子优化核酸序列(SEQ ID NO:41)均由苏州工业园区唯可达生物科技有限公司提供。设计引物(见表9)，以pET-30a(+)-CTB(苏州工业园区唯可达生物科技有限公司)为模板，用PCR方法扩增含有CTB编码序列的核酸片段，具体方法参看Ex Taq酶(宝生物，货号RR001B)试剂说明书。然后通过同源重组的方式将该核酸片段插入pET-30a(+)-LMN载体上，构建成pET-30a(+)-LMN-CTB载体(质粒图谱如图11)，经测序鉴定正确后入库。用限制内切酶Nco I和Xho I鉴定载体pET-30a(+)-LMN-CTB(酶切体系如表10)，其酶切验证图谱如图12所示。
表9：实施例5中的引物
Figure BDA0001815105190000121
表10：实施例5中的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-LMN-CTB 3μL，约1μg
Nco I(宝生物，货号1160A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH<sub>2</sub>O 补至10μL
实施例6含有LMN-CTB与CMV Th表位融合蛋白的原核表达载体构建
从表4中选取10个来源于CMV的Th表位pp65-11、pp65-71、pp65-92、pp65-123、pp65-128、pp65-57、pp65-62、pp65-30、pp65-112和pp65-104，将其串联在一起，所组成的氨基酸序列如SEQ ID NO:44所示，其中，该SEQ ID NO:44中的一段序列“EFELRRQ”是因为引入了酶切位点导致的，属于融合构建常用的技术。通过在线密码子优化软件(http://www.jcat.de/)将所述Th表位氨基酸序列优化成大肠杆菌密码子使用偏好的核苷酸序列(SEQ ID NO:45)，经上海捷瑞生物技术有限公司合成后，通过本领域熟知的分子生物学方法插入至原核表达载体pET-30a(+)(Novagen，货号69909)上的多克隆位点Nco I与Xho I之间，构建成可表达融合蛋白抗原的原核表达构建体pET-30a(+)-CMV Th(质粒图谱如图13)，经测序鉴定正确后入库。用限制内切酶Mlu I和Xho I鉴定载体pET-30a(+)-CMV Th(酶切体系如表11)，其酶切验证图谱如图14所示。
其中，如图13所示，CMV Th1含有5个CMV的Th表位，由pp65-11、pp65-71、pp65-92、pp65-123和pp65-128串联而成，CMV Th2含有5个CMV的Th表位，由pp65-57、pp65-62、pp65-30、pp65-112和pp65-104组成，CMV Th1和CMV Th2之间引入了EcoR I、Sac I和Sal I等3个限制酶酶切位点。
表11：质粒pET-30a(+)-CMV Th的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-CMV Th 3μL，约1μg
Mlu I(宝生物，货号1071A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH<sub>2</sub>O 补至10μL
设计引物(见表12)，以实施例5中的pET-30a(+)-LMN-CTB为模板，用PCR方法扩增含有LMN-CTB编码序列的核酸片段，具体方法参看Ex Taq酶(宝生物，货号RR001B)试剂说明书。然后通过本领域熟知的分子生物学方法将该核酸片段插入实施例6中的pET-30a(+)-CMV Th载体上的Not I和Xho I之间，构建成pET-30a(+)-CMV10-LMNB载体(质粒图谱如图15，经测序鉴定正确后入库。用限制内切酶BamH I和Xho I鉴定载体pET-30a(+)-CMV10-LMNB(酶切体系如表13)，其酶切验证图谱如图16所示。如图15所示，pET-30a(+)-CMV10-LMNB含有CMV Th1和CMV Th2片段，即该载体含有表4中所有10个CMV Th表位。这些表位是pp65-11、pp65-71、pp65-92、pp65-123、pp65-128、pp65-57、pp65-62、pp65-30、pp65-112和pp65-104。
表12：实施例6中的引物设计
引物 序列
6F(SEQ ID NO:46) GCGCGGCCGCGACGACAAGGCCATGGCT
6R(SEQ ID NO:47) GCCTCGAGGTTAGCCATAGAGATAGC
表13：pET-30a(+)-CMV10-LMNB的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-CMV10-LMNB 3μL，约1μg
BamH I(宝生物，货号1010A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH<sub>2</sub>O 补至10μL
实施例7含有LMN-CTB与Influ Th表位融合蛋白的原核表达载体构建
从表4中选取13个来源于流感病毒的Th表位HA203、NP438、NS1-84、M1-181、HA375、NP24、NP95、NP221、HA434、HA440、NP324、M1-127和M1-210，将其串联在一起，所组成的氨基酸序列如SEQ ID NO:48所示。
通过在线密码子优化软件(http://www.jcat.de/)将所述含有流感病毒Th表位的氨基酸序列优化成大肠杆菌密码子使用偏好的核苷酸序列(SEQ ID NO:49)，经上海捷瑞生物技术有限公司合成后，通过本领域熟知的分子生物学方法插入至原核表达载体pET-30a(+)(Novagen，货号69909)上的多克隆位点Nco I与Xho I之间，构建成可表达融合蛋白抗原的原核表达构建体pET-30a(+)-Influ Th(质粒图谱如图17)，经测序鉴定正确后入库。用限制内切酶Nco I和Xho I鉴定载体pET-30a(+)-Influ Th(酶切体系如表14)，其酶切验证图谱如图18所示。
其中，如图17所示，Influ Th1含有8个流感病毒的Th表位，由HA203、NP438、NS1-84、M1-181、HA375、NP24、NP95和NP221串联而成，Influ Th2含有5个流感病毒的Th表位，由HA434、HA440、NP324、M1-127和M1-210组成，Influ Th1和Influ Th2之间引入了EcoR I、SacI和Sal I等3个限制酶酶切位点。
表14：实施例7中的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-Influ Th 3μL，约1μg
Nco I(宝生物，货号1160A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH<sub>2</sub>O 补至10μL
设计引物(见表15)，以实施例5中的pET-30a(+)-LMN-CTB为模板，用PCR方法扩增含有LMN-CTB编码序列的核酸片段，具体方法参看Ex Taq酶(宝生物，货号RR001B)试剂说明书。然后通过本领域熟知的分子生物学方法将该核酸片段插入实施例7中的pET-30a(+)-Influ Th载体上的Not I和Sal I之间，构建成pET-30a(+)-Influ8-LMNB载体(含8个流感病毒Th表位，质粒图谱如图19)，经测序鉴定正确后入库。用限制内切酶BamH I和Xho I鉴定载体pET-30a(+)-Influ8-LMNB(酶切体系如表16)，其酶切验证图谱如图20所示。
如图19所示，pET-30a(+)-Influ8-LMNB载体含有Influ Th1片段，即含有包括表4中的HA203、NP438、NS1-84、M1-181、HA375、NP24、NP95和NP221在内的8个流感病毒Th表位。
表15：实施例7中的引物
引物 序列
7F1(SEQ ID NO:50) GCGCGGCCGCGTTAGCCATAGAGATAGC
7R1(SEQ ID NO:51) GCGTCGACAAGACGACAAGGCCATGGCTATGC
表16：质粒pET-30a(+)-Influ8-LMNB的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-Influ8-LMNB 3μL，约1μg
BamH I(宝生物，货号1010A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH<sub>2</sub>O 补至10μL
设计引物(见表17)，以实施例5中的pET-30a(+)-LMN-CTB为模板，用PCR方法扩增含有LMN-CTB编码序列的核酸片段，具体方法参看Ex Taq酶(宝生物，货号RR001B)试剂说明书。然后通过本领域熟知的分子生物学方法将该核酸片段分别插入实施例6中的pET-30a(+)-Influ Th载体上的Not I和Xho I之间，构建成pET-30a(+)-Influ13-LMNB载体(含13个流感病毒Th表位，质粒图谱如图21)，经测序鉴定正确后入库。用限制内切酶BamH I和Xho I鉴定载体pET-30a(+)-CMV10-LMNB(酶切体系如表18)，其酶切验证图谱如图22所示。
如图21所示，pET-30a(+)-Influ13-LMNB载体含有Influ Th1和Influ Th2片段，即含有包括表4中的HA203、NP438、NS1-84、M1-181、HA375、NP24、NP95和NP221在内的8个流感病毒Th表位和包括表4中的HA434、HA440、NP324、M1-127和M1-210在内的5个流感病毒Th表位。该载体总共包括表4中的所有13个流感病毒Th表位。
表17：实施例7中的引物设计
引物 序列
7F2(SEQ ID NO:52) GCCTCGAGGTTAGCCATAGAGATAGCA
7R2(SEQ ID NO:53) GCGCGGCCGCGACGACAAGGCCATGGCTATG
表18：实施例7中的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-Influ13-LMNB 3μL，约1μg
BamH I(宝生物，货号1010A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH<sub>2</sub>O 补至10μL
实施例8融合蛋白的表达纯化
分别将实施例4中构建的原核表达载体pET-30a(+)-LMN、实施例5中构建的原核表达载体pET-30a(+)-LMN-CTB、实施例6中构建的原核表达载体pET-30a(+)-CMV5-LMNB和pET-30a(+)-CMV10-LMNB、实施例7中构建的原核表达载体pET-30a(+)-Influ8-LMNB和pET-30a(+)-Influ13-LMNB转化至BL21(DE3)感受态细胞(天根生化科技(北京)有限公司，货号CB105，转化方法参见感受态细胞说明书)，根据《pET系统手册》(TB055 8th Edition02/99，Novagen)分别制备重组蛋白LMN(其氨基酸序列如SEQ ID NO:59所示)、LMNB(其氨基酸序列如SEQ ID NO:54所示)、LMNB-C10(其氨基酸序列如SEQ ID NO:58所示)、LMNB-I8(其氨基酸序列如SEQ ID NO:55所示)和LMNB-13(其氨基酸序列如SEQ ID NO:56所示)，所制蛋白分装后，-80℃保存。
经BCA法(碧云天生物技术研究所，货号P0009)检测，所制备的重组蛋白浓度为1mg/mL，检测方法参看检测试剂盒说明书。经凝胶法(厦门鲎试剂实验厂有限公司，货号G011000)检测，所制备的重组蛋白内毒素含量<1EU/mg，符合动物实验要求，检测方法参看鲎试剂说明书。
实施例9动物免疫实验
实施例2、3以及实施例8所制备的疫苗信息如表19。DNA疫苗载体pVKD1.0由苏州工业园区唯可达公司提供，DNA疫苗pVKD1.0-NP(表达源于毒株A/Shanghai/02/2013(H7N9))的流感抗原NP(NCBI参考序列:YP_009118476.1)由苏州工业园区唯可达公司提供，蛋白疫苗VP1(肠道病毒71型VP1蛋白，参见中国专利申请201310088364.5)由苏州工业园区唯可达公司提供。
从苏州大学动物实验中心购买16只6-8周龄的雌性BAL B/c小鼠，并饲养于苏州大学动物实验中心SPF级动物房中。实验动物分组与疫苗接种规划如表20。所有DNA疫苗均为小腿胫骨前肌肉注射，100μg/只。所有蛋白疫苗均在与完全弗氏佐剂(CFA)或不完全弗氏佐剂(IFA)充分乳化后，背部皮下注射，10μg/只。最后一次免疫完成后两周，处死小鼠，收集血清和脾细胞，分别进行酶联免疫斑点实验(ELISPOT)和酶联免疫实验(ELISA)。
小鼠IFN-γELISPOT试剂盒购自美国BD公司(货号：551083)，方法参见BD公司IFN-γELISPOT试剂盒说明书。刺激肽为NY-ESO-1 41#肽(WITQCFLPVFLAQPP)，为上海科肽生物科技有限公司合成，刺激终浓度为10μg/mL。阳性刺激物佛波醇-12-肉豆蔻酯-13-乙酯(phorbol-12-myristate-13-acetate,PMA)和离子霉素(inomysin)均购自美国Sigma公司。
ELISA的方法为本领域人员熟知，简述如下。96孔酶标板购自江海玻璃仪器总厂。重组LMN和NY-ESO-1均由苏州工业园区唯可达生物科技有限公司提供。用NaHCO3缓冲液(pH9.6)包被蛋白，4℃过夜，包被浓度为10μg/mL。用含0.1％牛血清白蛋白(bovine serumalbumin,BSA)的磷酸盐缓冲液(PBS)37℃封闭30分钟，再用含0.5％吐温20的磷酸盐缓冲液(PBST)洗5遍后，室温孵育小鼠血清1小时，初始稀释度为1:100，PBST洗5遍后，1:5000孵育羊抗鼠HRP二抗(美国Santacruz公司)，37℃、30分钟，PBST洗5遍后用3,3,5,5四甲基联苯胺(3，3，5，5-tetraamthyl benzidine,TMB)底物显色，37℃、15分钟，用2M稀硫酸终止后用酶标仪(美国Thermo公司)在450nm波长下读取吸光度(A)值。大于阴性对照A值2.1倍判断为阳性值，阳性值中的最高稀释度的倒数定义为血清抗体滴度。滴度小于起始稀释度1:100时，定义其滴度为50。
表19：疫苗信息
Figure BDA0001815105190000181
表20：分组与免疫规划
Figure BDA0001815105190000182
细胞免疫应答检测结果如图23所示。其中，初免pVKD1.0-CI-LMNB DNA疫苗，LMNB-I13蛋白加强(即实施例8中的D组)免疫效果最好，显著性高于其平行对照(B组)以及LMNB-I8加强组(C组))。而且LMNB-I13蛋白加强组的细胞免疫应答水平与平行对照组(B组)相比，提高了将近3倍。表明装有流感病毒13个Th表位(D组)能大幅度提高弱免疫原的细胞免疫应答水平。
实施例10含有LMN-CTB与CMV Th表位融合蛋白的原核表达载体构建
设计引物(见表21)，以实施例5中的pET-30a(+)-LMN-CTB为模板，用PCR方法扩增含有LMN-CTB编码序列的核酸片段，具体方法参看Ex Taq酶(宝生物，货号RR001B)试剂说明书。然后通过本领域熟知的分子生物学方法将该核酸片段插入实施例6中的pET-30a(+)-CMV Th载体上的Not I和Sal I之间，构建成pET-30a(+)-CMV5-LMNB载体(质粒图谱如图24)，经测序鉴定正确后入库。用限制内切酶BamH I和Xho I鉴定载体pET-30a(+)-CMV5-LMNB(酶切体系如表22)，其酶切验证图谱如图25所示。如图24所示，pET-30a(+)-CMV5-LMNB含有CMV Th1片段，即该载体含有表4中前5个CMV Th表位。这些表位是pp65-11、pp65-71、pp65-92、pp65-123和pp65-128。
表21：实施例10中的引物设计
引物 序列
7F1(SEQ ID NO:50) GCGCGGCCGCGTTAGCCATAGAGATAGC
7R1(SEQ ID NO:51) GCGTCGACAAGACGACAAGGCCATGGCTATGC
表22：pET-30a(+)-CMV10-LMNB的酶切鉴定体系(37℃酶切过夜)
酶切体系 体积
质粒pET-30a(+)-CMV10-LMNB 3μL，约1μg
BamH I(宝生物，货号1010A) 1μL
Xho I(宝生物，货号1094A) 1μL
酶切缓冲液 1μL
ddH<sub>2</sub>O 补至10μL
实施例11融合蛋白的表达纯化
如实施例8所述，将实施例10中构建的原核表达载体pET-30a(+)-CMV5-LMNB转化至BL21(DE3)感受态细胞(天根生化科技(北京)有限公司，货号CB105，转化方法参见感受态细胞说明书)，根据《pET系统手册》(TB055 8th Edition02/99，Novagen)制备重组蛋白LMNB-C5(其氨基酸序列如SEQ ID NO:57所示)，所制蛋白分装后，-80℃保存。
经BCA法(碧云天生物技术研究所，货号P0009)检测，所制备的重组蛋白浓度为1mg/mL，检测方法参看检测试剂盒说明书。经凝胶法(厦门鲎试剂实验厂有限公司，货号G011000)检测，所制备的重组蛋白内毒素含量<1EU/mg，符合动物实验要求，检测方法参看鲎试剂说明书。
实施例12动物免疫实验
疫苗信息如表19。DNA疫苗pVKD1.0-CI(实施例3)由苏州工业园区唯可达公司提供。
从苏州大学动物实验中心购买20只6-8周龄的雌性BAL B/c小鼠，并饲养于苏州大学动物实验中心SPF级动物房中。实验动物分组与疫苗接种规划如表23。所有DNA疫苗均为小腿胫骨前肌肉注射，100μg/只。所有蛋白疫苗均在与完全弗氏佐剂(CFA)或不完全弗氏佐剂(IFA)充分乳化后，背部皮下注射，10μg/只。最后一次免疫完成后两周，处死小鼠，收集血清和脾细胞，分别进行酶联免疫斑点实验(ELISPOT)和酶联免疫实验(ELISA)。
小鼠IFN-γELISPOT试剂盒购自美国BD公司(货号：551083)，方法参见BD公司IFN-γELISPOT试剂盒说明书。刺激肽为NY-ESO-1 41#肽(WITQCFLPVFLAQPP)，为上海科肽生物科技有限公司合成，刺激终浓度为10μg/mL。阳性刺激物佛波醇-12-肉豆蔻酯-13-乙酯(phorbol-12-myristate-13-acetate,PMA)和离子霉素(inomysin)均购自美国Sigma公司。
ELISA的方法为本领域人员熟知，简述如下。96孔酶标板购自江海玻璃仪器总厂。重组LMN和NY-ESO-1均由苏州工业园区唯可达生物科技有限公司提供。用NaHCO3缓冲液(pH9.6)包被蛋白，4℃过夜，包被浓度为10μg/mL。用含0.1％牛血清白蛋白(bovine serumalbumin,BSA)的磷酸盐缓冲液(PBS)37℃封闭30分钟，再用含0.5％吐温20的磷酸盐缓冲液(PBST)洗5遍后，室温孵育小鼠血清1小时，初始稀释度为1:100，PBST洗5遍后，1:5000孵育羊抗鼠HRP二抗(美国Santacruz公司)，37℃、30分钟，PBST洗5遍后用3,3,5,5四甲基联苯胺(3，3，5，5-tetraamthyl benzidine,TMB)底物显色，37℃、15分钟，用2M稀硫酸终止后用酶标仪(美国Thermo公司)在450nm波长下读取吸光度(A)值。大于阴性对照A值2.1倍判断为阳性值，阳性值中的最高稀释度的倒数定义为血清抗体滴度。滴度小于起始稀释度1:100时，定义其滴度为50。
表23：分组与免疫规划
Figure BDA0001815105190000211
细胞免疫应答检测结果如图26所示。其中，初免pVKD1.0-CI-LMNB DNA疫苗，LMNB-C5蛋白加强(即实施例11中的C组)和LMNB-C10蛋白加强(即实施例11中的D组)免疫效果最好，显著性高于其平行对照(B组)。表明装有CMV病毒5个Th表位(C组)和10个Th表位(D组)均能大幅度提高弱免疫原的细胞免疫应答水平。
实施例13肿瘤预防动物实验
实施例2、3以及实施例8所制备的疫苗信息如表19。DNA疫苗载体pVKD1.0由苏州工业园区唯可达公司提供，DNA疫苗pVKD1.0-NP(表达源于毒株A/Shanghai/02/2013(H7N9))的流感抗原NP(NCBI参考序列:YP_009118476.1)由苏州工业园区唯可达生物科技有限公司提供，蛋白疫苗VP1(肠道病毒71型VP1蛋白，参见中国专利申请201310088364.5)由苏州工业园区唯可达生物科技有限公司提供。
从苏州大学动物实验中心购买60只6-8周龄的雌性BAL B/c小鼠，并饲养于苏州大学动物实验中心SPF级动物房中。实验动物分组与疫苗接种规划如表24。所有DNA疫苗均为小腿胫骨前肌肉注射，100μg/只。所有蛋白疫苗均在与完全弗氏佐剂(CFA)或不完全弗氏佐剂(IFA)充分乳化后，背部皮下注射，10μg/只。最后一次免疫完成后两周，小鼠皮下接种4T1-hNY-ESO-1稳定转染细胞系(由苏州工业园区唯可达生物科技有限公司提供)，接种剂量为1×105细胞/只，接种后连续观察并测量肿瘤生长情况。按照以下公式计算肿瘤体积：肿瘤体积(mm3)＝长×宽2/2。当小鼠肿瘤体积超过2000mm3时，对小鼠处死。
表24：分组与免疫规划
Figure BDA0001815105190000221
各组免疫小鼠肿瘤生长情况如图27所示。其中，对照组(A组)小鼠在攻瘤后(即肿瘤接种后)第14天全部出现肿瘤，并迅速生长。各免疫组小鼠肿瘤生长滞后于对照组，其中，LMNB-I13加强免疫组(D组)小鼠和LMNB-I13与LMNB-C10混合加强免疫组(E组)小鼠肿瘤生长最慢，因此，此两组疫苗效果最好。
此外，还进行了小鼠无瘤存活分析，结果如图28所示。其中，对照组A组小鼠的无瘤存活(TFS)中位数为14天。各疫苗免疫组小鼠无瘤存活均显著高于对照组。表明所有疫苗均能提高免疫后小鼠的无瘤存活期。其中，带有I13表位融合肽的D组，带有I13和C10表位融合肽的E组和F组效果最好，最高增加了一倍的小鼠无瘤存活期。与常规疫苗组(B组)相比，带有I13表位融合肽的疫苗组(D组)显著提高了无瘤存活期，提高了约40％的无瘤存活。表明装有流感病毒13个Th表位或10个CMV Th表位能大幅提高肿瘤疫苗的肿瘤保护效果。
最后，还进行了小鼠总体存活分析，结果如图29所示。其中，对照组A组小鼠的总体存活(OS)中位数为35天。各疫苗免疫组小鼠总体存活均显著高于对照组，表明所有疫苗均能提高免疫后小鼠的存活。其中，带有I13表位融合肽的D组，带有I13和C10表位融合肽的E组和F组效果最好，总体存活最高增加了83％。与常规疫苗组(B组)相比，带有I13表位融合肽的疫苗组(D组和F组)显著增加了无瘤存活期，最高增加了28％，表明装有流感病毒13个Th表位或10个CMV Th表位能大幅提高肿瘤疫苗的肿瘤保护效果。
实施例14肿瘤治疗实验
所涉及的疫苗见实施例9。从苏州大学动物实验中心购买30只6-8周龄的雌性BALB/c小鼠，并饲养于苏州大学动物实验中心SPF级动物房中。实验动物分组与疫苗接种规划如表25。所有DNA疫苗均为小腿胫骨前肌肉注射，100μg/只。所有蛋白疫苗均在与完全弗氏佐剂(CFA)或不完全弗氏佐剂(IFA)充分乳化后，背部皮下注射，10μg/只。最后一次免疫完成后两周，小鼠皮下接种肿瘤细胞4T1-hNY-ESO-1稳定转染细胞系(由苏州工业园区唯可达生物科技有限公司提供)，接种剂量为1×105细胞/只，分别在肿瘤细胞接种后第1天，第8天和第15天给相应小鼠皮下接种蛋白疫苗。接种后连续观察并测量肿瘤生长情况。按照以下公式计算肿瘤体积：肿瘤体积(mm3)＝长×宽2/2。当小鼠肿瘤体积超过2000mm3时，对小鼠处死。
表25：分组与免疫规划
Figure BDA0001815105190000231
各组免疫小鼠肿瘤生长情况如图30所示。其中，对照组(A组)小鼠在攻瘤后(即肿瘤接种后)第14天全部出现肿瘤，并迅速生长。与未治疗的对照组(A组)相比，LMNB-I13蛋白疫苗治疗组(C组)小鼠肿瘤生长最慢。而且，在小鼠攻瘤后第22天，LMNB-I13蛋白疫苗治疗组小鼠肿瘤大小显著小于对照组(A组)，直到第30天，两组之间的肿瘤大小还存在显著性差异。到第35天，C组小鼠肿瘤生长开始加快，可能与LMNB-I13蛋白疫苗停止接种有关。该结果表明LMNB-I13蛋白疫苗能抑制小鼠肿瘤生长。
实施例15肿瘤治疗实验
所涉及的疫苗见实施例9。从苏州大学动物实验中心购买30只6-8周龄的雌性BALB/c小鼠，并饲养于苏州大学动物实验中心SPF级动物房中。实验动物分组与疫苗接种规划如表26。所有DNA疫苗均为小腿胫骨前肌肉注射，100μg/只。所有蛋白疫苗均在与完全弗氏佐剂(CFA)或不完全弗氏佐剂(IFA)充分乳化后，背部皮下注射，10μg/只。最后一次免疫完成后两周，小鼠皮下接种肿瘤细胞CT26-hLAGE-1稳定转染细胞系(由苏州工业园区唯可达生物科技有限公司提供)，接种剂量为1×105细胞/只，分别在肿瘤细胞接种后第1天，第8天和第15天给相应小鼠皮下接种蛋白疫苗。接种后连续观察并测量肿瘤生长情况。按照以下公式计算肿瘤体积：肿瘤体积(mm3)＝长×宽2/2。当小鼠肿瘤体积超过2000mm3时，对小鼠处死。
表26：分组与免疫规划
Figure BDA0001815105190000241
各组免疫小鼠肿瘤生长情况如图31所示，由于未治疗对照组(A组)小鼠在攻瘤后(即肿瘤接种后)有小鼠未能成功接种肿瘤，故未纳入分析，比较了平行疫苗对照组(B组)和LMNB-I13治疗组(C组)小鼠。与B相比，C组小鼠肿瘤生长较慢，而且在在小鼠攻瘤后第22天，LMNB-I13蛋白疫苗治疗组小鼠肿瘤大小显著小于平行疫苗对照组(B组)，直到第30天，两组之间的肿瘤大小还存在显著性差异。同样地，在CT26小鼠模型也观察到C组小鼠在后期肿瘤生长开始加快，可能与LMNB-I13蛋白疫苗停止接种有关。该结果表明LMNB-I13蛋白疫苗能抑制小鼠肿瘤生长。
序列表
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<213> 人工序列(Artificial Sequence)
<400> 18
Arg Met Cys Asn Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg
1 5 10 15
Ala Met
<210> 19
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg
1 5 10 15
Thr
<210> 20
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp
1 5 10 15
Ser
<210> 21
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
His Lys Ser Gln Leu Val Trp Met Ala Cys Asn Ser Ala Ala Phe Glu
1 5 10 15
Asp
<210> 22
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Cys Met Gly Leu Ile Tyr Asn Arg Met Gly Ala Val Thr Thr Glu Ser
1 5 10 15
Ala
<210> 23
<211> 23
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Arg Gln Met Val Gln Ala Met Arg Ala Ile Gly Thr His Pro Ser Ser
1 5 10 15
Ser Thr Gly Leu Lys Asn Asp
20
<210> 24
<211> 210
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Met Gln Ala Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp
1 5 10 15
Gly Pro Gly Gly Pro Gly Ile Pro Asp Gly Pro Gly Gly Asn Ala Gly
20 25 30
Gly Pro Gly Glu Ala Gly Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala
35 40 45
Gly Ala Ala Arg Ala Ser Gly Pro Arg Gly Gly Ala Pro Arg Gly Pro
50 55 60
His Gly Gly Ala Ala Ser Ala Gln Asp Gly Arg Cys Pro Cys Gly Ala
65 70 75 80
Arg Arg Pro Asp Ser Arg Leu Leu Glu Leu His Ile Thr Met Pro Phe
85 90 95
Ser Ser Pro Met Glu Ala Glu Leu Val Arg Arg Ile Leu Ser Arg Asp
100 105 110
Ala Ala Pro Leu Pro Arg Pro Gly Ala Val Leu Lys Asp Phe Thr Val
115 120 125
Ser Gly Asn Leu Leu Phe Met Ser Val Arg Asp Gln Asp Arg Glu Gly
130 135 140
Ala Gly Arg Met Arg Val Val Gly Trp Gly Leu Gly Ser Ala Ser Pro
145 150 155 160
Glu Gly Gln Lys Ala Arg Asp Leu Arg Thr Pro Lys His Lys Val Ser
165 170 175
Glu Gln Arg Pro Gly Thr Pro Gly Pro Pro Pro Pro Glu Gly Ala Gln
180 185 190
Gly Asp Gly Cys Arg Gly Val Ala Phe Asn Val Met Phe Ser Ala Pro
195 200 205
His Ile
210
<210> 25
<211> 313
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Pro Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu Glu
1 5 10 15
Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala Thr
20 25 30
Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val Thr
35 40 45
Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser Pro
50 55 60
Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp Ser
65 70 75 80
Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser Thr
85 90 95
Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys Val
100 105 110
Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu Pro
115 120 125
Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln Tyr
130 135 140
Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu Val
145 150 155 160
Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr Ile
165 170 175
Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp Asn
180 185 190
Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile Ile
195 200 205
Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu Leu
210 215 220
Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly Asp
225 230 235 240
Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu Glu
245 250 255
Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu Trp
260 265 270
Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His His
275 280 285
Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu His
290 295 300
Glu Trp Val Leu Arg Glu Gly Glu Glu
305 310
<210> 26
<211> 179
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Gln Ala Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp Gly
1 5 10 15
Pro Gly Gly Pro Gly Ile Pro Asp Gly Pro Gly Gly Asn Ala Gly Gly
20 25 30
Pro Gly Glu Ala Gly Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala Gly
35 40 45
Ala Ala Arg Ala Ser Gly Pro Gly Gly Gly Ala Pro Arg Gly Pro His
50 55 60
Gly Gly Ala Ala Ser Gly Leu Asn Gly Cys Cys Arg Cys Gly Ala Arg
65 70 75 80
Gly Pro Glu Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe Ala
85 90 95
Thr Pro Met Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gln Asp Ala
100 105 110
Pro Pro Leu Pro Val Pro Gly Val Leu Leu Lys Glu Phe Thr Val Ser
115 120 125
Gly Asn Ile Leu Thr Ile Arg Leu Thr Ala Ala Asp His Arg Gln Leu
130 135 140
Gln Leu Ser Ile Ser Ser Cys Leu Gln Gln Leu Ser Leu Leu Met Trp
145 150 155 160
Ile Thr Gln Cys Phe Leu Pro Val Phe Leu Ala Gln Pro Pro Ser Gly
165 170 175
Gln Arg Arg
<210> 27
<211> 630
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
atgcaggccg aaggccgggg cacagggggt tcgacgggcg atgctgatgg cccaggaggc 60
cctggcattc ctgatggccc agggggcaat gctggcggcc caggagaggc gggtgccacg 120
ggcggcagag gtccccgggg cgcaggggca gcaagggcct cggggccgag aggaggcgcc 180
ccgcggggtc cgcatggcgg tgccgcttct gcgcaggatg gaaggtgccc ctgcggggcc 240
aggaggccgg acagccgcct gcttgagttg cacatcacga tgcctttctc gtcgccaatg 300
gaagcggagc tggtccgcag aatcctgtcc cgggatgccg caccgctccc ccgaccaggg 360
gcggttctga aggacttcac cgtgtccggc aacctactgt ttatgtcagt tcgggaccag 420
gacagggaag gcgctgggcg gatgagggtg gtgggttggg ggctgggatc agcctccccg 480
gaggggcaga aagctagaga tctcagaaca cccaaacaca aggtctcaga acagagacct 540
ggtacaccag gcccgccgcc acccgaggga gcccagggag atgggtgcag aggtgtcgcc 600
tttaatgtga tgttctctgc ccctcacatt 630
<210> 28
<211> 939
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
cccctggagc agcgcagcca gcactgcaag cccgaggagg gcctggaggc ccgcggcgag 60
gccctgggcc tggtgggcgc ccaggccccc gccaccgagg agcaggaggc cgccagcagc 120
agcagcaccc tggtggaggt gaccctgggc gaggtgcccg ccgccgagag ccccgacccc 180
ccccagagcc cccagggcgc cagcagcctg cccaccacca tgaactaccc cctgtggagc 240
cagagctacg aggacagcag caaccaggag gaggagggcc ccagcacctt ccccgacctg 300
gagagcgagt tccaggccgc cctgagccgc aaggtggccg agctggtgca cttcctgctg 360
ctgaagtacc gcgcccgcga gcccgtgacc aaggccgaga tgctgggcag cgtggtgggc 420
aactggcagt acttcttccc cgtgatcttc agcaaggcca gcagcagcct gcagctggtg 480
ttcggcatcg agctgatgga ggtggacccc atcggccacc tgtacatctt cgccacctgc 540
ctgggcctga gctacgacgg cctgctgggc gacaaccaga tcatgcccaa ggccggcctg 600
ctgatcatcg tgctggccat catcgcccgc gagggcgact gcgcccccga ggagaagatc 660
tgggaggagc tgagcgtgct ggaggtgttc gagggccgcg aggacagcat cctgggcgac 720
cccaagaagc tgctgaccca gcacttcgtg caggagaact acctggagta ccgccaggtg 780
cccggcagcg accccgcctg ctacgagttc ctgtggggcc cccgcgccct ggtggagacc 840
agctacgtga aggtgctgca ccacatggtg aagatcagcg gcggccccca catcagctac 900
ccccccctgc acgagtgggt gctgcgcgag ggcgaggag 939
<210> 29
<211> 537
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
caggccgaag gccggggcac agggggttcg acgggcgatg ctgatggccc aggaggccct 60
ggcattcctg atggcccagg gggcaatgct ggcggcccag gagaggcggg tgccacgggc 120
ggcagaggtc cccggggcgc aggggcagca agggcctcgg ggccgggagg aggcgccccg 180
cggggtccgc atggcggcgc ggcttcaggg ctgaatggat gctgcagatg cggggccagg 240
gggccggaga gccgcctgct tgagttctac ctcgccatgc ctttcgcgac acccatggaa 300
gcagagctgg cccgcaggag cctggcccag gatgccccac cgcttcccgt gccaggggtg 360
cttctgaagg agttcactgt gtccggcaac atactgacta tccgactgac tgctgcagac 420
caccgccaac tgcagctctc catcagctcc tgtctccagc agctttccct gttgatgtgg 480
atcacgcagt gctttctgcc cgtgtttttg gctcagcctc cctcagggca gaggcgc 537
<210> 30
<211> 123
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Ile Lys Leu Lys Phe Gly Val Phe Phe Thr Val Leu Leu Ser Ser Ala
1 5 10 15
Tyr Ala His Gly Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Tyr
20 25 30
His Asn Thr Gln Ile His Thr Leu Asn Asp Lys Ile Phe Ser Tyr Thr
35 40 45
Glu Ser Leu Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn
50 55 60
Gly Ala Thr Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser
65 70 75 80
Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr
85 90 95
Leu Thr Glu Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr
100 105 110
Pro His Ala Ile Ala Ala Ile Ser Met Ala Asn
115 120
<210> 31
<211> 369
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
atcaagctga agttcggcgt gttcttcacc gtgctgctga gcagcgccta cgcccacggc 60
accccccaga acatcaccga cctgtgcgcc gagtaccaca acacccagat ccacaccctg 120
aacgacaaga tcttcagcta caccgagagc ctggccggca agcgcgagat ggccatcatc 180
accttcaaga acggcgccac cttccaggtg gaggtgcccg gcagccagca catcgacagc 240
cagaagaagg ccatcgagcg catgaaggac accctgcgca tcgcctacct gaccgaggcc 300
aaggtggaga agctgtgcgt gtggaacaac aagacccccc acgccatcgc cgccatcagc 360
atggccaac 369
<210> 32
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
tccctcaggg cagaggcgca tcaagctgaa gttcggcgtg 40
<210> 33
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
gaaggcacag cagatctgga tcctcagttg gccatgctga tggc 44
<210> 34
<211> 219
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 34
Met Leu Leu Gln Thr Gly Ile His Val Arg Val Ser Gln Pro Ser Leu
1 5 10 15
Ile Ile Lys Pro Gly Lys Ile Ser His Ile Met Leu Asp Val Ala Glu
20 25 30
His Pro Thr Phe Thr Ser Gln Tyr Arg Ile Gln Gly Lys Leu Ala Gly
35 40 45
Ile Leu Ala Arg Asn Leu Val Pro Met Val Ala Thr Val Lys Tyr Gln
50 55 60
Glu Phe Phe Trp Asp Ala Asn Asp Ile Tyr Arg Ile Asn Gln Arg Ala
65 70 75 80
Leu Tyr His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser Asp Met
85 90 95
Arg Ala Glu Ile Ile Lys Met Met Glu Ser Ala Arg Pro Glu Ala Leu
100 105 110
Ala Ser Arg Tyr Leu Thr Asp Met Thr Ile Glu Glu Met Ser Arg Leu
115 120 125
Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met Ala Gly Ser Ser Glu
130 135 140
Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly
145 150 155 160
Ile Thr Asn Lys Val Asn Glu Ile Arg Ala Ser Val Gly Lys Met Ile
165 170 175
Asp Gly Ile Gly Arg Phe Tyr Ile Pro Ile Tyr Arg Arg Val Asp Gly
180 185 190
Lys Trp Met Arg Glu Leu Val Leu Tyr Arg Met Cys Asn Ile Leu Lys
195 200 205
Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met
210 215
<210> 35
<211> 657
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
atgctgctgc aaaccggcat ccacgtgcgc gtgagccagc ccagcctgat catcaagccc 60
ggcaagatca gccacatcat gctggacgtg gccgagcacc ccaccttcac cagccagtac 120
cgcatccagg gcaagctggc cggcatcctg gcccgcaacc tggtgcccat ggtggccacc 180
gtgaagtacc aggagttctt ctgggacgcc aacgacatct accgcatcaa ccagcgcgcc 240
ctgtaccaca ccgagaacgc ctacgtgagc gtggtgagca gcgacatgcg cgccgagatc 300
atcaagatga tggagagcgc ccgccccgag gccctggcca gccgctacct gaccgacatg 360
accatcgagg agatgagccg cctggccagc accaccgcca aggccatgga gcagatggcc 420
ggcagcagcg agagcggcta cgccgccgac cagaagagca cccagaacgc catcaacggc 480
atcaccaaca aggtgaacga gatccgcgcc agcgtgggca agatgatcga cggcatcggc 540
cgcttctaca tccccatcta ccgccgcgtg gacggcaagt ggatgcgcga gctggtgctg 600
taccgcatgt gcaacatcct gaagggcaag ttccagaccg ccgcccagcg cgccatg 657
<210> 36
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
gcgcggccgc tgtcaccgtc gtcgacatgc aggccgaa 38
<210> 37
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
gcgatcctca gttggccatg ctgatggcgg cgatg 35
<210> 38
<211> 630
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
atgcaggctg aaggtcgtgg taccggtggt tctaccggtg acgctgacgg tccgggtggt 60
ccgggtatcc cggacggtcc gggtggtaac gctggtggtc cgggtgaagc tggtgctacc 120
ggtggtcgtg gtccgcgtgg tgctggtgct gctcgtgctt ctggtccgcg tggtggtgct 180
ccgcgtggtc cgcacggtgg tgctgcttct gctcaggacg gtcgttgccc gtgcggtgct 240
cgtcgtccgg actctcgtct gctggaactg cacatcacca tgccgttctc ttctccgatg 300
gaagctgaac tggttcgtcg tatcctgtct cgtgacgctg ctccgctgcc gcgtccgggt 360
gctgttctga aagacttcac cgtttctggt aacctgctgt tcatgtctgt tcgtgaccag 420
gaccgtgaag gtgctggtcg tatgcgtgtt gttggttggg gtctgggttc tgcttctccg 480
gaaggtcaga aagctcgtga cctgcgtacc ccgaaacaca aagtttctga acagcgtccg 540
ggtaccccgg gtccgccgcc gccggaaggt gctcagggtg acggttgccg tggtgttgct 600
ttcaacgtta tgttctctgc tccgcacatc 630
<210> 39
<211> 939
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
ccgctggaac agcgttctca gcactgcaaa ccggaagaag gtctggaagc tcgtggtgaa 60
gctctgggtc tggttggtgc tcaggctccg gctaccgaag aacaggaagc tgcttcttct 120
tcttctaccc tggttgaagt taccctgggt gaagttccgg ctgctgaatc tccggacccg 180
ccgcagtctc cgcagggtgc ttcttctctg ccgaccacca tgaactaccc gctgtggtct 240
cagtcttacg aagactcttc taaccaggaa gaagaaggtc cgtctacctt cccggacctg 300
gaatctgaat tccaggctgc tctgtctcgt aaagttgctg aactggttca cttcctgctg 360
ctgaaatacc gtgctcgtga accggttacc aaagctgaaa tgctgggttc tgttgttggt 420
aactggcagt acttcttccc ggttatcttc tctaaagctt cttcttctct gcagctggtt 480
ttcggtatcg aactgatgga agttgacccg atcggtcacc tgtacatctt cgctacctgc 540
ctgggtctgt cttacgacgg tctgctgggt gacaaccaga tcatgccgaa agctggtctg 600
ctgatcatcg ttctggctat catcgctcgt gaaggtgact gcgctccgga agaaaaaatc 660
tgggaagaac tgtctgttct ggaagttttc gaaggtcgtg aagactctat cctgggtgac 720
ccgaaaaaac tgctgaccca gcacttcgtt caggaaaact acctggaata ccgtcaggtt 780
ccgggttctg acccggcttg ctacgaattc ctgtggggtc cgcgtgctct ggttgaaacc 840
tcttacgtta aagttctgca ccacatggtt aaaatctctg gtggtccgca catctcttac 900
ccgccgctgc acgaatgggt tctgcgtgaa ggtgaagaa 939
<210> 40
<211> 540
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
gctcaggctg aaggtcgtgg taccggtggt tctaccggtg acgctgacgg tccgggtggt 60
ccgggtatcc cggacggtcc gggtggtaac gctggtggtc cgggtgaagc tggtgctacc 120
ggtggtcgtg gtccgcgtgg tgctggtgct gctcgtgctt ctggtccggg tggtggtgct 180
ccgcgtggtc cgcacggtgg tgctgcttct ggtctgaacg gttgctgccg ttgcggtgct 240
cgtggtccgg aatctcgtct gctggaattc tacctggcta tgccgttcgc taccccgatg 300
gaagctgaac tggctcgtcg ttctctggct caggacgctc cgccgctgcc ggttccgggt 360
gttctgctga aagaattcac cgtttctggt aacatcctga ccatccgtct gaccgctgct 420
gaccaccgtc agctgcagct gtctatctct tcttgcctgc agcagctgtc tctgctgatg 480
tggatcaccc agtgcttcct gccggttttc ctggctcagc cgccgtctgg tcagcgtcgt 540
<210> 41
<211> 369
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
atcaaactga aatttggcgt cttcttcacc gtcctgctgt cttctgctta cgctcacggt 60
accccgcaga acatcaccga cctgtgcgct gaataccaca acacccagat ccacaccctg 120
aacgacaaaa tcttctctta caccgaatct ctggctggta aacgtgaaat ggctatcatc 180
accttcaaaa acggtgctac cttccaggtt gaagttccgg gttctcagca catcgactct 240
cagaaaaaag ctatcgaacg tatgaaagac accctgcgta tcgcttacct gaccgaagct 300
aaagttgaaa aactgtgcgt ttggaacaac aaaaccccgc acgctatcgc tgctatctct 360
atggctaac 369
<210> 42
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
ggtggtggtg gtgctcgagt tagttagcca tagaga 36
<210> 43
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
tctgcgtgaa ggtgaagaag ctcaggctga aggtcgtgg 39
<210> 44
<211> 157
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 44
Leu Leu Gln Thr Gly Ile His Val Arg Val Ser Gln Pro Ser Leu Ile
1 5 10 15
Ile Lys Pro Gly Lys Ile Ser His Ile Met Leu Asp Val Ala Glu His
20 25 30
Pro Thr Phe Thr Ser Gln Tyr Arg Ile Gln Gly Lys Leu Ala Gly Ile
35 40 45
Leu Ala Arg Asn Leu Val Pro Met Val Ala Thr Val Lys Tyr Gln Glu
50 55 60
Phe Phe Trp Asp Ala Asn Asp Ile Tyr Arg Ile Glu Phe Glu Leu Arg
65 70 75 80
Arg Gln Lys Val Tyr Leu Glu Ser Phe Cys Glu Asp Val Pro Ser Gly
85 90 95
Lys Thr Leu Gly Ser Asp Val Glu Glu Asp Leu Thr Met Thr Arg Asn
100 105 110
Pro Leu Lys Met Leu Asn Ile Pro Ser Ile Asn Val His His Tyr Ala
115 120 125
Cys Thr Ser Gly Val Met Thr Arg Gly Arg Leu Lys Ala Glu Thr Glu
130 135 140
Arg Lys Thr Pro Arg Val Thr Gly Gly Gly Ala Met Ala
145 150 155
<210> 45
<211> 471
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
ctgctgcaga ccggtatcca cgttcgtgtt tctcagccgt ctctgatcat caaaccgggt 60
aaaatctctc acatcatgct ggacgttgct gaacacccga ccttcacctc tcagtaccgt 120
atccagggta aactggctgg tatcctggct cgtaacctgg ttccgatggt tgctaccgtt 180
aaataccagg aattcttctg ggacgctaac gacatctacc gtatcgaatt cgagctccgt 240
cgacaaaaag tttacctgga atctttctgc gaagacgttc cgtctggtaa aaccctgggt 300
tctgacgttg aagaagacct gaccatgacc cgtaacccgc tgaaaatgct gaacatcccg 360
tctatcaacg ttcaccacta cgcttgcacc tctggtgtta tgacccgtgg tcgtctgaaa 420
gctgaaaccg aacgtaaaac cccgcgtgtt accggtggtg gtgctatggc t 471
<210> 46
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
gcgcggccgc gacgacaagg ccatggct 28
<210> 47
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
gcctcgaggt tagccataga gatagc 26
<210> 48
<211> 230
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 48
Asn Gln Arg Ala Leu Tyr His Thr Glu Asn Ala Tyr Val Ser Val Val
1 5 10 15
Ser Ser Asp Met Arg Ala Glu Ile Ile Lys Met Met Glu Ser Ala Arg
20 25 30
Pro Glu Ala Leu Ala Ser Arg Tyr Leu Thr Asp Met Thr Ile Glu Glu
35 40 45
Met Ser Arg Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met Ala
50 55 60
Gly Ser Ser Glu Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn
65 70 75 80
Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Glu Ile Arg Ala Ser Val
85 90 95
Gly Lys Met Ile Asp Gly Ile Gly Arg Phe Tyr Ile Pro Ile Tyr Arg
100 105 110
Arg Val Asp Gly Lys Trp Met Arg Glu Leu Val Leu Tyr Arg Met Cys
115 120 125
Asn Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Glu
130 135 140
Phe Glu Leu Arg Arg Gln Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val
145 150 155 160
Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser His Lys Ser
165 170 175
Gln Leu Val Trp Met Ala Cys Asn Ser Ala Ala Phe Glu Asp Cys Met
180 185 190
Gly Leu Ile Tyr Asn Arg Met Gly Ala Val Thr Thr Glu Ser Ala Arg
195 200 205
Gln Met Val Gln Ala Met Arg Ala Ile Gly Thr His Pro Ser Ser Ser
210 215 220
Thr Gly Leu Lys Asn Asp
225 230
<210> 49
<211> 690
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
aaccagcgtg ctctgtacca caccgaaaac gcttacgttt cggttgtaag ctctgacatg 60
cgtgctgaaa tcatcaaaat gatggaatct gctcgtccgg aagctctggc ttctcgttac 120
ctgaccgaca tgaccatcga agaaatgtct cgtctggctt ctaccaccgc taaagctatg 180
gaacagatgg ctggttcttc tgaatctggt tacgctgctg accagaaatc tacccagaac 240
gctatcaacg gtatcaccaa caaagttaac gaaatccgtg cttctgttgg taaaatgatc 300
gacggtatag gcaggttcta catcccgata taccgtcgtg ttgacggtaa atggatgcgt 360
gaactggttc tgtaccgtat gtgcaacatc ctgaaaggta aattccagac cgctgctcag 420
cgtgctatgg aattcgagct ccgtcgacaa atctggacct acaacgctga actgctggtt 480
ctgctggaaa acgaacgtac cctggacttc cacgactctc acaaatctca gctggtttgg 540
atggcttgca actcggcggc gttcgaagac tgcatgggtc tgatctacaa ccgtatgggt 600
gctgttacca ccgaatctgc tcgtcagatg gttcaggcta tgcgtgctat cggtacccac 660
ccgtcttctt ctaccggtct gaaaaacgac 690
<210> 50
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
gcgcggccgc gttagccata gagatagc 28
<210> 51
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
gcgtcgacaa gacgacaagg ccatggctat gc 32
<210> 52
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
gcctcgaggt tagccataga gatagca 27
<210> 53
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
gcgcggccgc gacgacaagg ccatggctat g 31
<210> 54
<211> 872
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 54
Met His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser
1 5 10 15
Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp
20 25 30
Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Ala Met Gln
35 40 45
Ala Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp Gly Pro
50 55 60
Gly Gly Pro Gly Ile Pro Asp Gly Pro Gly Gly Asn Ala Gly Gly Pro
65 70 75 80
Gly Glu Ala Gly Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala Gly Ala
85 90 95
Ala Arg Ala Ser Gly Pro Arg Gly Gly Ala Pro Arg Gly Pro His Gly
100 105 110
Gly Ala Ala Ser Ala Gln Asp Gly Arg Cys Pro Cys Gly Ala Arg Arg
115 120 125
Pro Asp Ser Arg Leu Leu Glu Leu His Ile Thr Met Pro Phe Ser Ser
130 135 140
Pro Met Glu Ala Glu Leu Val Arg Arg Ile Leu Ser Arg Asp Ala Ala
145 150 155 160
Pro Leu Pro Arg Pro Gly Ala Val Leu Lys Asp Phe Thr Val Ser Gly
165 170 175
Asn Leu Leu Phe Met Ser Val Arg Asp Gln Asp Arg Glu Gly Ala Gly
180 185 190
Arg Met Arg Val Val Gly Trp Gly Leu Gly Ser Ala Ser Pro Glu Gly
195 200 205
Gln Lys Ala Arg Asp Leu Arg Thr Pro Lys His Lys Val Ser Glu Gln
210 215 220
Arg Pro Gly Thr Pro Gly Pro Pro Pro Pro Glu Gly Ala Gln Gly Asp
225 230 235 240
Gly Cys Arg Gly Val Ala Phe Asn Val Met Phe Ser Ala Pro His Ile
245 250 255
Pro Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu Glu
260 265 270
Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala Thr
275 280 285
Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val Thr
290 295 300
Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser Pro
305 310 315 320
Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp Ser
325 330 335
Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser Thr
340 345 350
Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys Val
355 360 365
Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu Pro
370 375 380
Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln Tyr
385 390 395 400
Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu Val
405 410 415
Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr Ile
420 425 430
Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp Asn
435 440 445
Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile Ile
450 455 460
Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu Leu
465 470 475 480
Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly Asp
485 490 495
Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu Glu
500 505 510
Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu Trp
515 520 525
Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His His
530 535 540
Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu His
545 550 555 560
Glu Trp Val Leu Arg Glu Gly Glu Glu Ala Gln Ala Glu Gly Arg Gly
565 570 575
Thr Gly Gly Ser Thr Gly Asp Ala Asp Gly Pro Gly Gly Pro Gly Ile
580 585 590
Pro Asp Gly Pro Gly Gly Asn Ala Gly Gly Pro Gly Glu Ala Gly Ala
595 600 605
Thr Gly Gly Arg Gly Pro Arg Gly Ala Gly Ala Ala Arg Ala Ser Gly
610 615 620
Pro Gly Gly Gly Ala Pro Arg Gly Pro His Gly Gly Ala Ala Ser Gly
625 630 635 640
Leu Asn Gly Cys Cys Arg Cys Gly Ala Arg Gly Pro Glu Ser Arg Leu
645 650 655
Leu Glu Phe Tyr Leu Ala Met Pro Phe Ala Thr Pro Met Glu Ala Glu
660 665 670
Leu Ala Arg Arg Ser Leu Ala Gln Asp Ala Pro Pro Leu Pro Val Pro
675 680 685
Gly Val Leu Leu Lys Glu Phe Thr Val Ser Gly Asn Ile Leu Thr Ile
690 695 700
Arg Leu Thr Ala Ala Asp His Arg Gln Leu Gln Leu Ser Ile Ser Ser
705 710 715 720
Cys Leu Gln Gln Leu Ser Leu Leu Met Trp Ile Thr Gln Cys Phe Leu
725 730 735
Pro Val Phe Leu Ala Gln Pro Pro Ser Gly Gln Arg Arg Ile Lys Leu
740 745 750
Lys Phe Gly Val Phe Phe Thr Val Leu Leu Ser Ser Ala Tyr Ala His
755 760 765
Gly Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Tyr His Asn Thr
770 775 780
Gln Ile His Thr Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu Ser Leu
785 790 795 800
Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly Ala Thr
805 810 815
Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys Lys
820 825 830
Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu
835 840 845
Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala
850 855 860
Ile Ala Ala Ile Ser Met Ala Asn
865 870
<210> 55
<211> 1043
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 55
Met His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser
1 5 10 15
Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp
20 25 30
Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Ala Asp Ile
35 40 45
Gly Ser Asn Gln Arg Ala Leu Tyr His Thr Glu Asn Ala Tyr Val Ser
50 55 60
Val Val Ser Ser Asp Met Arg Ala Glu Ile Ile Lys Met Met Glu Ser
65 70 75 80
Ala Arg Pro Glu Ala Leu Ala Ser Arg Tyr Leu Thr Asp Met Thr Ile
85 90 95
Glu Glu Met Ser Arg Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln
100 105 110
Met Ala Gly Ser Ser Glu Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr
115 120 125
Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Glu Ile Arg Ala
130 135 140
Ser Val Gly Lys Met Ile Asp Gly Ile Gly Arg Phe Tyr Ile Pro Ile
145 150 155 160
Tyr Arg Arg Val Asp Gly Lys Trp Met Arg Glu Leu Val Leu Tyr Arg
165 170 175
Met Cys Asn Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala
180 185 190
Met Glu Phe Glu Leu Arg Arg Gln Asp Asp Lys Ala Met Ala Met Gln
195 200 205
Ala Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp Gly Pro
210 215 220
Gly Gly Pro Gly Ile Pro Asp Gly Pro Gly Gly Asn Ala Gly Gly Pro
225 230 235 240
Gly Glu Ala Gly Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala Gly Ala
245 250 255
Ala Arg Ala Ser Gly Pro Arg Gly Gly Ala Pro Arg Gly Pro His Gly
260 265 270
Gly Ala Ala Ser Ala Gln Asp Gly Arg Cys Pro Cys Gly Ala Arg Arg
275 280 285
Pro Asp Ser Arg Leu Leu Glu Leu His Ile Thr Met Pro Phe Ser Ser
290 295 300
Pro Met Glu Ala Glu Leu Val Arg Arg Ile Leu Ser Arg Asp Ala Ala
305 310 315 320
Pro Leu Pro Arg Pro Gly Ala Val Leu Lys Asp Phe Thr Val Ser Gly
325 330 335
Asn Leu Leu Phe Met Ser Val Arg Asp Gln Asp Arg Glu Gly Ala Gly
340 345 350
Arg Met Arg Val Val Gly Trp Gly Leu Gly Ser Ala Ser Pro Glu Gly
355 360 365
Gln Lys Ala Arg Asp Leu Arg Thr Pro Lys His Lys Val Ser Glu Gln
370 375 380
Arg Pro Gly Thr Pro Gly Pro Pro Pro Pro Glu Gly Ala Gln Gly Asp
385 390 395 400
Gly Cys Arg Gly Val Ala Phe Asn Val Met Phe Ser Ala Pro His Ile
405 410 415
Pro Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu Glu
420 425 430
Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala Thr
435 440 445
Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val Thr
450 455 460
Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser Pro
465 470 475 480
Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp Ser
485 490 495
Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser Thr
500 505 510
Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys Val
515 520 525
Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu Pro
530 535 540
Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln Tyr
545 550 555 560
Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu Val
565 570 575
Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr Ile
580 585 590
Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp Asn
595 600 605
Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile Ile
610 615 620
Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu Leu
625 630 635 640
Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly Asp
645 650 655
Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu Glu
660 665 670
Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu Trp
675 680 685
Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His His
690 695 700
Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu His
705 710 715 720
Glu Trp Val Leu Arg Glu Gly Glu Glu Ala Gln Ala Glu Gly Arg Gly
725 730 735
Thr Gly Gly Ser Thr Gly Asp Ala Asp Gly Pro Gly Gly Pro Gly Ile
740 745 750
Pro Asp Gly Pro Gly Gly Asn Ala Gly Gly Pro Gly Glu Ala Gly Ala
755 760 765
Thr Gly Gly Arg Gly Pro Arg Gly Ala Gly Ala Ala Arg Ala Ser Gly
770 775 780
Pro Gly Gly Gly Ala Pro Arg Gly Pro His Gly Gly Ala Ala Ser Gly
785 790 795 800
Leu Asn Gly Cys Cys Arg Cys Gly Ala Arg Gly Pro Glu Ser Arg Leu
805 810 815
Leu Glu Phe Tyr Leu Ala Met Pro Phe Ala Thr Pro Met Glu Ala Glu
820 825 830
Leu Ala Arg Arg Ser Leu Ala Gln Asp Ala Pro Pro Leu Pro Val Pro
835 840 845
Gly Val Leu Leu Lys Glu Phe Thr Val Ser Gly Asn Ile Leu Thr Ile
850 855 860
Arg Leu Thr Ala Ala Asp His Arg Gln Leu Gln Leu Ser Ile Ser Ser
865 870 875 880
Cys Leu Gln Gln Leu Ser Leu Leu Met Trp Ile Thr Gln Cys Phe Leu
885 890 895
Pro Val Phe Leu Ala Gln Pro Pro Ser Gly Gln Arg Arg Ile Lys Leu
900 905 910
Lys Phe Gly Val Phe Phe Thr Val Leu Leu Ser Ser Ala Tyr Ala His
915 920 925
Gly Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Tyr His Asn Thr
930 935 940
Gln Ile His Thr Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu Ser Leu
945 950 955 960
Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly Ala Thr
965 970 975
Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys Lys
980 985 990
Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu
995 1000 1005
Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala
1010 1015 1020
Ile Ala Ala Ile Ser Met Ala Asn Ala Ala Ala Leu Glu His His His
1025 1030 1035 1040
His His His
<210> 56
<211> 1125
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 56
Met His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser
1 5 10 15
Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp
20 25 30
Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Ala Asp Ile
35 40 45
Gly Ser Asn Gln Arg Ala Leu Tyr His Thr Glu Asn Ala Tyr Val Ser
50 55 60
Val Val Ser Ser Asp Met Arg Ala Glu Ile Ile Lys Met Met Glu Ser
65 70 75 80
Ala Arg Pro Glu Ala Leu Ala Ser Arg Tyr Leu Thr Asp Met Thr Ile
85 90 95
Glu Glu Met Ser Arg Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln
100 105 110
Met Ala Gly Ser Ser Glu Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr
115 120 125
Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Glu Ile Arg Ala
130 135 140
Ser Val Gly Lys Met Ile Asp Gly Ile Gly Arg Phe Tyr Ile Pro Ile
145 150 155 160
Tyr Arg Arg Val Asp Gly Lys Trp Met Arg Glu Leu Val Leu Tyr Arg
165 170 175
Met Cys Asn Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala
180 185 190
Met Glu Phe Glu Leu Arg Arg Gln Ile Trp Thr Tyr Asn Ala Glu Leu
195 200 205
Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser His
210 215 220
Lys Ser Gln Leu Val Trp Met Ala Cys Asn Ser Ala Ala Phe Glu Asp
225 230 235 240
Cys Met Gly Leu Ile Tyr Asn Arg Met Gly Ala Val Thr Thr Glu Ser
245 250 255
Ala Arg Gln Met Val Gln Ala Met Arg Ala Ile Gly Thr His Pro Ser
260 265 270
Ser Ser Thr Gly Leu Lys Asn Asp Gln Ala Cys Gly Arg Asp Asp Lys
275 280 285
Ala Met Ala Met Gln Ala Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly
290 295 300
Asp Ala Asp Gly Pro Gly Gly Pro Gly Ile Pro Asp Gly Pro Gly Gly
305 310 315 320
Asn Ala Gly Gly Pro Gly Glu Ala Gly Ala Thr Gly Gly Arg Gly Pro
325 330 335
Arg Gly Ala Gly Ala Ala Arg Ala Ser Gly Pro Arg Gly Gly Ala Pro
340 345 350
Arg Gly Pro His Gly Gly Ala Ala Ser Ala Gln Asp Gly Arg Cys Pro
355 360 365
Cys Gly Ala Arg Arg Pro Asp Ser Arg Leu Leu Glu Leu His Ile Thr
370 375 380
Met Pro Phe Ser Ser Pro Met Glu Ala Glu Leu Val Arg Arg Ile Leu
385 390 395 400
Ser Arg Asp Ala Ala Pro Leu Pro Arg Pro Gly Ala Val Leu Lys Asp
405 410 415
Phe Thr Val Ser Gly Asn Leu Leu Phe Met Ser Val Arg Asp Gln Asp
420 425 430
Arg Glu Gly Ala Gly Arg Met Arg Val Val Gly Trp Gly Leu Gly Ser
435 440 445
Ala Ser Pro Glu Gly Gln Lys Ala Arg Asp Leu Arg Thr Pro Lys His
450 455 460
Lys Val Ser Glu Gln Arg Pro Gly Thr Pro Gly Pro Pro Pro Pro Glu
465 470 475 480
Gly Ala Gln Gly Asp Gly Cys Arg Gly Val Ala Phe Asn Val Met Phe
485 490 495
Ser Ala Pro His Ile Pro Leu Glu Gln Arg Ser Gln His Cys Lys Pro
500 505 510
Glu Glu Gly Leu Glu Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala
515 520 525
Gln Ala Pro Ala Thr Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr
530 535 540
Leu Val Glu Val Thr Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp
545 550 555 560
Pro Pro Gln Ser Pro Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn
565 570 575
Tyr Pro Leu Trp Ser Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu
580 585 590
Glu Gly Pro Ser Thr Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala
595 600 605
Leu Ser Arg Lys Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr
610 615 620
Arg Ala Arg Glu Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val Val
625 630 635 640
Gly Asn Trp Gln Tyr Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser
645 650 655
Ser Leu Gln Leu Val Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile
660 665 670
Gly His Leu Tyr Ile Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly
675 680 685
Leu Leu Gly Asp Asn Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile
690 695 700
Val Leu Ala Ile Ile Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys
705 710 715 720
Ile Trp Glu Glu Leu Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp
725 730 735
Ser Ile Leu Gly Asp Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln
740 745 750
Glu Asn Tyr Leu Glu Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys
755 760 765
Tyr Glu Phe Leu Trp Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val
770 775 780
Lys Val Leu His His Met Val Lys Ile Ser Gly Gly Pro His Ile Ser
785 790 795 800
Tyr Pro Pro Leu His Glu Trp Val Leu Arg Glu Gly Glu Glu Ala Gln
805 810 815
Ala Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp Gly Pro
820 825 830
Gly Gly Pro Gly Ile Pro Asp Gly Pro Gly Gly Asn Ala Gly Gly Pro
835 840 845
Gly Glu Ala Gly Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala Gly Ala
850 855 860
Ala Arg Ala Ser Gly Pro Gly Gly Gly Ala Pro Arg Gly Pro His Gly
865 870 875 880
Gly Ala Ala Ser Gly Leu Asn Gly Cys Cys Arg Cys Gly Ala Arg Gly
885 890 895
Pro Glu Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe Ala Thr
900 905 910
Pro Met Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gln Asp Ala Pro
915 920 925
Pro Leu Pro Val Pro Gly Val Leu Leu Lys Glu Phe Thr Val Ser Gly
930 935 940
Asn Ile Leu Thr Ile Arg Leu Thr Ala Ala Asp His Arg Gln Leu Gln
945 950 955 960
Leu Ser Ile Ser Ser Cys Leu Gln Gln Leu Ser Leu Leu Met Trp Ile
965 970 975
Thr Gln Cys Phe Leu Pro Val Phe Leu Ala Gln Pro Pro Ser Gly Gln
980 985 990
Arg Arg Ile Lys Leu Lys Phe Gly Val Phe Phe Thr Val Leu Leu Ser
995 1000 1005
Ser Ala Tyr Ala His Gly Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala
1010 1015 1020
Glu Tyr His Asn Thr Gln Ile His Thr Leu Asn Asp Lys Ile Phe Ser
1025 1030 1035 1040
Tyr Thr Glu Ser Leu Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe
1045 1050 1055
Lys Asn Gly Ala Thr Phe Gln Val Glu Val Pro Gly Ser Gln His Ile
1060 1065 1070
Asp Ser Gln Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile
1075 1080 1085
Ala Tyr Leu Thr Glu Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn
1090 1095 1100
Lys Thr Pro His Ala Ile Ala Ala Ile Ser Met Ala Asn Leu Glu His
1105 1110 1115 1120
His His His His His
1125
<210> 57
<211> 975
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 57
Met His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser
1 5 10 15
Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp
20 25 30
Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Ala Asp Ile
35 40 45
Gly Ser Leu Leu Gln Thr Gly Ile His Val Arg Val Ser Gln Pro Ser
50 55 60
Leu Ile Ile Lys Pro Gly Lys Ile Ser His Ile Met Leu Asp Val Ala
65 70 75 80
Glu His Pro Thr Phe Thr Ser Gln Tyr Arg Ile Gln Gly Lys Leu Ala
85 90 95
Gly Ile Leu Ala Arg Asn Leu Val Pro Met Val Ala Thr Val Lys Tyr
100 105 110
Gln Glu Phe Phe Trp Asp Ala Asn Asp Ile Tyr Arg Ile Glu Phe Glu
115 120 125
Leu Arg Arg Gln Asp Asp Lys Ala Met Ala Met Gln Ala Glu Gly Arg
130 135 140
Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp Gly Pro Gly Gly Pro Gly
145 150 155 160
Ile Pro Asp Gly Pro Gly Gly Asn Ala Gly Gly Pro Gly Glu Ala Gly
165 170 175
Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala Gly Ala Ala Arg Ala Ser
180 185 190
Gly Pro Arg Gly Gly Ala Pro Arg Gly Pro His Gly Gly Ala Ala Ser
195 200 205
Ala Gln Asp Gly Arg Cys Pro Cys Gly Ala Arg Arg Pro Asp Ser Arg
210 215 220
Leu Leu Glu Leu His Ile Thr Met Pro Phe Ser Ser Pro Met Glu Ala
225 230 235 240
Glu Leu Val Arg Arg Ile Leu Ser Arg Asp Ala Ala Pro Leu Pro Arg
245 250 255
Pro Gly Ala Val Leu Lys Asp Phe Thr Val Ser Gly Asn Leu Leu Phe
260 265 270
Met Ser Val Arg Asp Gln Asp Arg Glu Gly Ala Gly Arg Met Arg Val
275 280 285
Val Gly Trp Gly Leu Gly Ser Ala Ser Pro Glu Gly Gln Lys Ala Arg
290 295 300
Asp Leu Arg Thr Pro Lys His Lys Val Ser Glu Gln Arg Pro Gly Thr
305 310 315 320
Pro Gly Pro Pro Pro Pro Glu Gly Ala Gln Gly Asp Gly Cys Arg Gly
325 330 335
Val Ala Phe Asn Val Met Phe Ser Ala Pro His Ile Pro Leu Glu Gln
340 345 350
Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu Glu Ala Arg Gly Glu
355 360 365
Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala Thr Glu Glu Gln Glu
370 375 380
Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val Thr Leu Gly Glu Val
385 390 395 400
Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser Pro Gln Gly Ala Ser
405 410 415
Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp Ser Gln Ser Tyr Glu
420 425 430
Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser Thr Phe Pro Asp Leu
435 440 445
Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys Val Ala Glu Leu Val
450 455 460
His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu Pro Val Thr Lys Ala
465 470 475 480
Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln Tyr Phe Phe Pro Val
485 490 495
Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu Val Phe Gly Ile Glu
500 505 510
Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr Ile Phe Ala Thr Cys
515 520 525
Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp Asn Gln Ile Met Pro
530 535 540
Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile Ile Ala Arg Glu Gly
545 550 555 560
Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu Leu Ser Val Leu Glu
565 570 575
Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly Asp Pro Lys Lys Leu
580 585 590
Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu Glu Tyr Arg Gln Val
595 600 605
Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu Trp Gly Pro Arg Ala
610 615 620
Leu Val Glu Thr Ser Tyr Val Lys Val Leu His His Met Val Lys Ile
625 630 635 640
Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu His Glu Trp Val Leu
645 650 655
Arg Glu Gly Glu Glu Ala Gln Ala Glu Gly Arg Gly Thr Gly Gly Ser
660 665 670
Thr Gly Asp Ala Asp Gly Pro Gly Gly Pro Gly Ile Pro Asp Gly Pro
675 680 685
Gly Gly Asn Ala Gly Gly Pro Gly Glu Ala Gly Ala Thr Gly Gly Arg
690 695 700
Gly Pro Arg Gly Ala Gly Ala Ala Arg Ala Ser Gly Pro Gly Gly Gly
705 710 715 720
Ala Pro Arg Gly Pro His Gly Gly Ala Ala Ser Gly Leu Asn Gly Cys
725 730 735
Cys Arg Cys Gly Ala Arg Gly Pro Glu Ser Arg Leu Leu Glu Phe Tyr
740 745 750
Leu Ala Met Pro Phe Ala Thr Pro Met Glu Ala Glu Leu Ala Arg Arg
755 760 765
Ser Leu Ala Gln Asp Ala Pro Pro Leu Pro Val Pro Gly Val Leu Leu
770 775 780
Lys Glu Phe Thr Val Ser Gly Asn Ile Leu Thr Ile Arg Leu Thr Ala
785 790 795 800
Ala Asp His Arg Gln Leu Gln Leu Ser Ile Ser Ser Cys Leu Gln Gln
805 810 815
Leu Ser Leu Leu Met Trp Ile Thr Gln Cys Phe Leu Pro Val Phe Leu
820 825 830
Ala Gln Pro Pro Ser Gly Gln Arg Arg Ile Lys Leu Lys Phe Gly Val
835 840 845
Phe Phe Thr Val Leu Leu Ser Ser Ala Tyr Ala His Gly Thr Pro Gln
850 855 860
Asn Ile Thr Asp Leu Cys Ala Glu Tyr His Asn Thr Gln Ile His Thr
865 870 875 880
Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu Ser Leu Ala Gly Lys Arg
885 890 895
Glu Met Ala Ile Ile Thr Phe Lys Asn Gly Ala Thr Phe Gln Val Glu
900 905 910
Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys Lys Ala Ile Glu Arg
915 920 925
Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu Ala Lys Val Glu
930 935 940
Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala Ile Ala Ala Ile
945 950 955 960
Ser Met Ala Asn Ala Ala Ala Leu Glu His His His His His His
965 970 975
<210> 58
<211> 1052
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 58
Met His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser
1 5 10 15
Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp
20 25 30
Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Ala Asp Ile
35 40 45
Gly Ser Leu Leu Gln Thr Gly Ile His Val Arg Val Ser Gln Pro Ser
50 55 60
Leu Ile Ile Lys Pro Gly Lys Ile Ser His Ile Met Leu Asp Val Ala
65 70 75 80
Glu His Pro Thr Phe Thr Ser Gln Tyr Arg Ile Gln Gly Lys Leu Ala
85 90 95
Gly Ile Leu Ala Arg Asn Leu Val Pro Met Val Ala Thr Val Lys Tyr
100 105 110
Gln Glu Phe Phe Trp Asp Ala Asn Asp Ile Tyr Arg Ile Glu Phe Glu
115 120 125
Leu Arg Arg Gln Lys Val Tyr Leu Glu Ser Phe Cys Glu Asp Val Pro
130 135 140
Ser Gly Lys Thr Leu Gly Ser Asp Val Glu Glu Asp Leu Thr Met Thr
145 150 155 160
Arg Asn Pro Leu Lys Met Leu Asn Ile Pro Ser Ile Asn Val His His
165 170 175
Tyr Ala Cys Thr Ser Gly Val Met Thr Arg Gly Arg Leu Lys Ala Glu
180 185 190
Thr Glu Arg Lys Thr Pro Arg Val Thr Gly Gly Gly Ala Met Ala Gln
195 200 205
Ala Cys Gly Arg Asp Asp Lys Ala Met Ala Met Gln Ala Glu Gly Arg
210 215 220
Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp Gly Pro Gly Gly Pro Gly
225 230 235 240
Ile Pro Asp Gly Pro Gly Gly Asn Ala Gly Gly Pro Gly Glu Ala Gly
245 250 255
Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala Gly Ala Ala Arg Ala Ser
260 265 270
Gly Pro Arg Gly Gly Ala Pro Arg Gly Pro His Gly Gly Ala Ala Ser
275 280 285
Ala Gln Asp Gly Arg Cys Pro Cys Gly Ala Arg Arg Pro Asp Ser Arg
290 295 300
Leu Leu Glu Leu His Ile Thr Met Pro Phe Ser Ser Pro Met Glu Ala
305 310 315 320
Glu Leu Val Arg Arg Ile Leu Ser Arg Asp Ala Ala Pro Leu Pro Arg
325 330 335
Pro Gly Ala Val Leu Lys Asp Phe Thr Val Ser Gly Asn Leu Leu Phe
340 345 350
Met Ser Val Arg Asp Gln Asp Arg Glu Gly Ala Gly Arg Met Arg Val
355 360 365
Val Gly Trp Gly Leu Gly Ser Ala Ser Pro Glu Gly Gln Lys Ala Arg
370 375 380
Asp Leu Arg Thr Pro Lys His Lys Val Ser Glu Gln Arg Pro Gly Thr
385 390 395 400
Pro Gly Pro Pro Pro Pro Glu Gly Ala Gln Gly Asp Gly Cys Arg Gly
405 410 415
Val Ala Phe Asn Val Met Phe Ser Ala Pro His Ile Pro Leu Glu Gln
420 425 430
Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu Glu Ala Arg Gly Glu
435 440 445
Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala Thr Glu Glu Gln Glu
450 455 460
Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val Thr Leu Gly Glu Val
465 470 475 480
Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser Pro Gln Gly Ala Ser
485 490 495
Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp Ser Gln Ser Tyr Glu
500 505 510
Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser Thr Phe Pro Asp Leu
515 520 525
Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys Val Ala Glu Leu Val
530 535 540
His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu Pro Val Thr Lys Ala
545 550 555 560
Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln Tyr Phe Phe Pro Val
565 570 575
Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu Val Phe Gly Ile Glu
580 585 590
Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr Ile Phe Ala Thr Cys
595 600 605
Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp Asn Gln Ile Met Pro
610 615 620
Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile Ile Ala Arg Glu Gly
625 630 635 640
Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu Leu Ser Val Leu Glu
645 650 655
Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly Asp Pro Lys Lys Leu
660 665 670
Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu Glu Tyr Arg Gln Val
675 680 685
Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu Trp Gly Pro Arg Ala
690 695 700
Leu Val Glu Thr Ser Tyr Val Lys Val Leu His His Met Val Lys Ile
705 710 715 720
Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu His Glu Trp Val Leu
725 730 735
Arg Glu Gly Glu Glu Ala Gln Ala Glu Gly Arg Gly Thr Gly Gly Ser
740 745 750
Thr Gly Asp Ala Asp Gly Pro Gly Gly Pro Gly Ile Pro Asp Gly Pro
755 760 765
Gly Gly Asn Ala Gly Gly Pro Gly Glu Ala Gly Ala Thr Gly Gly Arg
770 775 780
Gly Pro Arg Gly Ala Gly Ala Ala Arg Ala Ser Gly Pro Gly Gly Gly
785 790 795 800
Ala Pro Arg Gly Pro His Gly Gly Ala Ala Ser Gly Leu Asn Gly Cys
805 810 815
Cys Arg Cys Gly Ala Arg Gly Pro Glu Ser Arg Leu Leu Glu Phe Tyr
820 825 830
Leu Ala Met Pro Phe Ala Thr Pro Met Glu Ala Glu Leu Ala Arg Arg
835 840 845
Ser Leu Ala Gln Asp Ala Pro Pro Leu Pro Val Pro Gly Val Leu Leu
850 855 860
Lys Glu Phe Thr Val Ser Gly Asn Ile Leu Thr Ile Arg Leu Thr Ala
865 870 875 880
Ala Asp His Arg Gln Leu Gln Leu Ser Ile Ser Ser Cys Leu Gln Gln
885 890 895
Leu Ser Leu Leu Met Trp Ile Thr Gln Cys Phe Leu Pro Val Phe Leu
900 905 910
Ala Gln Pro Pro Ser Gly Gln Arg Arg Ile Lys Leu Lys Phe Gly Val
915 920 925
Phe Phe Thr Val Leu Leu Ser Ser Ala Tyr Ala His Gly Thr Pro Gln
930 935 940
Asn Ile Thr Asp Leu Cys Ala Glu Tyr His Asn Thr Gln Ile His Thr
945 950 955 960
Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu Ser Leu Ala Gly Lys Arg
965 970 975
Glu Met Ala Ile Ile Thr Phe Lys Asn Gly Ala Thr Phe Gln Val Glu
980 985 990
Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys Lys Ala Ile Glu Arg
995 1000 1005
Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu Ala Lys Val Glu
1010 1015 1020
Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala Ile Ala Ala Ile
1025 1030 1035 1040
Ser Met Ala Asn Leu Glu His His His His His His
1045 1050
<210> 59
<211> 757
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 59
Met His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser
1 5 10 15
Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp
20 25 30
Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Ala Met Gln
35 40 45
Ala Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp Gly Pro
50 55 60
Gly Gly Pro Gly Ile Pro Asp Gly Pro Gly Gly Asn Ala Gly Gly Pro
65 70 75 80
Gly Glu Ala Gly Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala Gly Ala
85 90 95
Ala Arg Ala Ser Gly Pro Arg Gly Gly Ala Pro Arg Gly Pro His Gly
100 105 110
Gly Ala Ala Ser Ala Gln Asp Gly Arg Cys Pro Cys Gly Ala Arg Arg
115 120 125
Pro Asp Ser Arg Leu Leu Glu Leu His Ile Thr Met Pro Phe Ser Ser
130 135 140
Pro Met Glu Ala Glu Leu Val Arg Arg Ile Leu Ser Arg Asp Ala Ala
145 150 155 160
Pro Leu Pro Arg Pro Gly Ala Val Leu Lys Asp Phe Thr Val Ser Gly
165 170 175
Asn Leu Leu Phe Met Ser Val Arg Asp Gln Asp Arg Glu Gly Ala Gly
180 185 190
Arg Met Arg Val Val Gly Trp Gly Leu Gly Ser Ala Ser Pro Glu Gly
195 200 205
Gln Lys Ala Arg Asp Leu Arg Thr Pro Lys His Lys Val Ser Glu Gln
210 215 220
Arg Pro Gly Thr Pro Gly Pro Pro Pro Pro Glu Gly Ala Gln Gly Asp
225 230 235 240
Gly Cys Arg Gly Val Ala Phe Asn Val Met Phe Ser Ala Pro His Ile
245 250 255
Pro Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu Glu
260 265 270
Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala Thr
275 280 285
Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val Thr
290 295 300
Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser Pro
305 310 315 320
Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp Ser
325 330 335
Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser Thr
340 345 350
Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys Val
355 360 365
Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu Pro
370 375 380
Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln Tyr
385 390 395 400
Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu Val
405 410 415
Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr Ile
420 425 430
Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp Asn
435 440 445
Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile Ile
450 455 460
Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu Leu
465 470 475 480
Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly Asp
485 490 495
Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu Glu
500 505 510
Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu Trp
515 520 525
Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His His
530 535 540
Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu His
545 550 555 560
Glu Trp Val Leu Arg Glu Gly Glu Glu Ala Gln Ala Glu Gly Arg Gly
565 570 575
Thr Gly Gly Ser Thr Gly Asp Ala Asp Gly Pro Gly Gly Pro Gly Ile
580 585 590
Pro Asp Gly Pro Gly Gly Asn Ala Gly Gly Pro Gly Glu Ala Gly Ala
595 600 605
Thr Gly Gly Arg Gly Pro Arg Gly Ala Gly Ala Ala Arg Ala Ser Gly
610 615 620
Pro Gly Gly Gly Ala Pro Arg Gly Pro His Gly Gly Ala Ala Ser Gly
625 630 635 640
Leu Asn Gly Cys Cys Arg Cys Gly Ala Arg Gly Pro Glu Ser Arg Leu
645 650 655
Leu Glu Phe Tyr Leu Ala Met Pro Phe Ala Thr Pro Met Glu Ala Glu
660 665 670
Leu Ala Arg Arg Ser Leu Ala Gln Asp Ala Pro Pro Leu Pro Val Pro
675 680 685
Gly Val Leu Leu Lys Glu Phe Thr Val Ser Gly Asn Ile Leu Thr Ile
690 695 700
Arg Leu Thr Ala Ala Asp His Arg Gln Leu Gln Leu Ser Ile Ser Ser
705 710 715 720
Cys Leu Gln Gln Leu Ser Leu Leu Met Trp Ile Thr Gln Cys Phe Leu
725 730 735
Pro Val Phe Leu Ala Gln Pro Pro Ser Gly Gln Arg Arg Leu Glu His
740 745 750
His His His His His
755

Claims (17)

1.一种CD4辅助性T细胞表位融合肽，其中所述表位融合肽为SEQ ID NO: 48所示的表位融合肽。
2.根据权利要求1所述的表位融合肽，其中所述表位融合肽诱导体液或细胞免疫应答。
3.一种融合蛋白，其包含权利要求1或2所述的表位融合肽和目的免疫原；
所述目的免疫原选自肿瘤抗原；所述肿瘤抗原选自LAGE抗原、MAGE抗原或NY-ESO-1抗原中的一种或多种。
4.根据权利要求3所述的融合蛋白，其中所述LAGE抗原为LAGE-1，所述MAGE抗原为MAGE-A3。
5.根据权利要求4所述的融合蛋白，其中所述LAGE-1的氨基酸序列如SEQ ID NO: 24所示，所述MAGE-A3的氨基酸序列如SEQ ID NO: 25所示，所述NY-ESO-1的氨基酸序列如SEQID NO: 26所示。
6.根据权利要求3所述的融合蛋白，其中所述肿瘤抗原为LAGE-1、MAGE-A3和NY-ESO-1。
7.根据权利要求3所述的融合蛋白，其中所述融合蛋白的氨基酸序列如SEQ ID NO: 56所示。
8.一种多核苷酸，其编码权利要求1或2所述的表位融合肽。
9.一种多核苷酸，其编码权利要求3-7任一项所述的融合蛋白。
10.一种免疫组合物，其包含预防或治疗有效量的权利要求1或2所述的表位融合肽，以及药学上可接受的载体。
11.一种免疫组合物，其包含预防或治疗有效量的权利要求3-7任一项所述的融合蛋白，以及药学上可接受的载体。
12.根据权利要求10所述的免疫组合物，其为疫苗。
13.根据权利要求11所述的免疫组合物，其为疫苗。
14.一种药盒，其包含权利要求1或2所述的表位融合肽、权利要求3-7任一项所述的融合蛋白和/或权利要求10-13任一项所述的免疫组合物，以及其使用说明。
15.权利要求1或2所述的表位融合肽、权利要求8所述的多核苷酸和/或权利要求10或12所述的免疫组合物在制备提高目的免疫原的免疫原性的药物中的用途；
所述目的免疫原选自肿瘤抗原；所述肿瘤抗原选自LAGE抗原、MAGE抗原或NY-ESO-1抗原中的一种或多种。
16.权利要求3-7任一项所述的融合蛋白、权利要求9所述的多核苷酸和/或权利要求11或13所述的免疫组合物在制备治疗或预防有需要的受试者的病症的药物中的用途；
所述有需要的受试者的病症选自恶性肿瘤。
17.根据权利要求16所述的用途，所述恶性肿瘤为乳腺癌或结肠癌。
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